摘要:

ENERGY TRANSFER IN SYSTEMS OF CONNECTED ORGANIC MOLECULES BY A. TERENIN, E. PUTZEIKO AND I. AKIMOV Optical Institute, The University, Leningrad, U.S.S.R. Received 12th January, 1959 Transport of charges, liberated by light in crystalline pigments (chlorophyll, methyl- chlorophyllide, haemin, the phthalocyanines) has been studied and their sign determined with two condenser methods. The positive carriers of photoconduction imply an oxida- tion-reduction intermolecular exchange of electron at the ground levels. Sensitization of the photoelectric effect in inorganic semiconductors by adsorbed pigments has been studied and interpreted as energy transfer. A light-induced proton exchange in layers of amphoteric acridine derivatives has been observed in fluorescence at low temperature, as an extension of a previous work on acridine in a matrix of organic acids in vacuo.Of the many aspects of energy migration in systems of organic molecules connected by ionic forces, hydrogen bonds, or van der Waals’ attraction, the intermolecular charge transfer presents one of the possibilities. The conduction in organic substances has formed in recent years the subject of so many in- vestigations, that it is impossible to review them here. We concentrated our attention on the transport of charges released by light in layers of tetrapyrrolic pigments related to chlorophyll, and this paper belongs to the sequence of re- searches in this direction.1-6 Another aspect of the problem is a proton transfer induced by light in a composite layer of acidic-basic organic components, which has been studied here before.7 There remains also the well-known possibility of an exciton migration at the singlet levels of a molecular system and in addition a migration of energy at its triplet level, the latter requiring due consideration, according to our last study.8 The transport of charges through microcrystalline dyes has been extensively studied in this laboratory by two methods.Vartanian studied the d.c. dark and d.c. transverse photoconductivity of layers of many dyes, deposited from solu- tions, or sublimed on to a gap between metallic electrodes.1.39 4 9 - 1 2 He obtained very valuable information on the thermal activation energies for the dark con- ductance, as well as for the photo-conductance. This method is dealing with the slow increase and decay of a direct current.Putzeiko 2 . 5 , 6 made extensive use of the condenser method of Bergmann 13 and also of the transverse photo-con- ductivity in the pigment layers, observed with intermittent light.5~ 6 The con- denser method has been supplemented by the determination of the sign of the charge carriers and the other method has allowed measurements of the relaxation times of the fast component of the photoconductivity. Recently another method, that of the vibrating condenser, has been applied by one of us (Akimov) to the same purpose. In it the change of the contact potential at the surface of a pigment layer was measured upon illumination by non-intermittent light. The results of this method corroborate in most cases the signs of the charge carriers, obtained in the Bergmann condenser.EXPERIMENTAL In the condenser method, described in our previous paper 14 a thin layer (02-0.5 mm) of the organic pigment in the form either of a microcrystalline powder, or of a sublimated, or a deposited layer from a solution, is inserted between the plates of a condenser, being 8384 ENERGY TRANSFER I N ORGANIC SYSTEMS prevented from metallic contact with them by dielectric laminae (0.1-0-2 mm). The layer is illuminated with monochromatic intermittent light (50-500 c/sec) through the entrance semi-transparent platinum coating of the condenser. The alternating current of the released charge carriers diffusing along the path of the light, owing to the concentration gradient, induces an alternating e.m.f.on the condenser, which after suitable amplification (gain x 100,OOO) and rectification, is measured as function of the wavelength of the in- cident monochromatic light (prism * and grating monochromators f5). The plots of the spectral curves of the photo-e.m.f. t in relative units are reduced to equal incident energy. The sign of the diffusing charge carriers can be obtained in the same set-up by com- pensating the photo-e.m.f. in the condenser by an additionally applied e.m.f. of a regulated magnitude and direction, synchronized with the former. This compensating e.m.f. was generated by a photocell illuminated by a beam of light split off from the incident light. FIG. 1 .-Vibrating condenser for measurements of the contact potential change of organic pigments under illumination.A, amplifier ; PD, phase-sensitive detector ; PI, phase inverter ; AO, audio oscillator ; EM, electromagnet ; M, monochromator. For an independent determination of the sign of the charge carriers, the method of the vibrating condenser has been used.$ The set-up consisted of a semi-transparent platinized mica lamina vibrating at a frequency of 140 c/sec, the other electrode being formed by the platinized flat wall of a glass trap, on which the pigment layer was deposited and which could be heated, or cooled from the inside (fig. 1). The distance between the electrodes was ca. 1 mm. The alternating potential due to the contact potential between the surface of the pigment and the platinized mica lamina was amplified by a narrow- band amplifier (gain x 25,000 to lOO,OOO), equipped with a phase-sensitive detector at the exit.A reference signal was impressed on the latter by the phase inverter from the same audio-oscillator which monitored the electromagnet of the vibrating electrode. The contact potential was compensated with a potentiometer, all the amplifying device working as sensitive null instrument which allowed us to measure changes of 0.05mV in contact potential on illumination of the pigment layer by monochromatic light.§ A change under illumination of the contact potential of the layer to more positive values was taken as indication of a diffusion of mobile negative charges inwards, and the reverse * Spectral band pass from 10 to 1 mp and incident energy from 10-3 to 10-5 W cm-2 t We use further the abbreviation " ph-e.m.f." for this method.1 It has been recognized that the Bergmann condenser method can give reliable results 9 Spectral band pass from 5 mp (at 700 mp) to 0.6 mp (at 400 mp) ; incident energy at 700 and 400 mp respectively. for a single type of charge carriers only. 9 x 10-5 to 3 x 10-7 W cm-2, respectively.A . TERENIN, E. PUTZEIKO A N D I . AKIMOV 85 change was ascribed to mobile positive charges released by light. This criterion has been checked in the same set-up and found to be confirmed by many photo-semiconductors with charge carriers of known sign (ZnO, TlI, CdS). The relaxation times (in the range 10-2-10-5 sec) for the transverse photoconductivity of the pigment layer under intermittent illumination (50-150 c/sec) have been measured with the oscillographic 7-meter of Feofilov and Tolstoi,ls which allows either a linear or an exponential sweep of the decay curve against time.The constant potential applied to the dye layer deposited on a gap ca. 1 mm wide between gold electrodes was 100 V. In this case only light-filters were used to limit the spectral range of the incident light. In all the methods, measurements have been performed in air and in vacuo, after de- gassing and thermal treatment, when admissible. RESULTS AND DISCUSSION SIGN OF THE CHARGE CARRIERS IN DYES AND PIGMENTS The signs of the charge carriers, obtained by the experimental method described above for various dyes and pigments in the form of microcrystalline powders, have been previously summarized in table 1 of ref.(14). Layers of the pigments were deposited from organic solutions or were sublimed (the phthalocyanines). 6 N E 3 E 4 I 0 w 3- E +A a 7 0 ' 2 I I I I I I 400 SO0 600 700 eoo $90 w FIG. 2.-Spectral dependence of contact potential change (A c. pot.) under illumination (reduced to equal incident energy) for a haemin layer ca. 1 p thick on Au. Curve 1, in air ; curve 2, in vacua The ordinate axis of the right half has scale values 10 times less. For most dyes the sign has been measured in air. In several cases the sign has been measured also in vacuo with the same result. Many dyes and in particular all the phthalocyanines (metal-free, or with Ag+, Cu2+, Mg2+, Zn2+, Co2+, Fez+, Fe3+) exhibit positive charge carriers of the ph-e.m.f. in the condenser. Negative charge carriers have been found in crystal violet.86 ENRGY TRANSFER I N ORGANIC SYSTEMS malachite green, pyoctanine, methyl eosin B, rhodamine B, pinacyanol.* For many dyes the Bergmann condenser method does not give a measurable ph-e.m.f. either in vacuo, or in air. To complement table l,14 where methylene blue, indigo, erythrosin, eosin, all the azo-dyes were given as photoelectrically insensitive, a ph-e.m.f. has not been observed also for the following dyes: brilliant green, thionin, indigo carmin, iodeosin, tropeolin 00, crocein and haemiatin. This does not imply that all these dyes do not possess any photoconductivity, but only that either they do not have a fast decaying current component, or that the charge carriers are of both signs, and in nearly equal amount, a circumstance in which I I 800 9 0 0 FIG.3.-Spectral response of photo-e.m.f. (reduced to equal incident energy). Curve 1, in microcrystalline powder of chlorophyll (a + b) in vacuo ; curve 2, in microcrystalline powder of methylchlorophyllide (a + b) in vacuo ; curve 3, methylchlorophyllide layer freshly deposited from a concentrated CHC13 solution. the condenser method fails. Thus, according to Vartanian, erythrosin and haemin exhibit an inertial d.c. photoconductivity of measurable magnitude. The vibrat- ing condenser did not show any change of contact potential on illumination of erythrosin, which is consistent with charge carriers of both signs. In haemin, positive holes have been detected by this method with a sensitivity spectrum shown in fig, 2, coinciding with that obtained in the d.c.method (Vartanian). In continuation of our work 5 ~ 6 on the photosemiconduction of chlorophyll, its derivatives and related tetrapyrollic pigments we have found that crystalline chlorophyll (a + b) and methylchlorophyllide (a + b) exhibit a marked e.m.f. in the condenser as microcrystalline dry powders on a dielectric support. Up to that time we obtained the ph-e.m.f. for chlorophyll and for ethylchlorophyllide only in the form of thin layers deposited on Pt from solutions and additionally treated by water vapour.6 We realize now that the metallic support and the accessory treatment were required to assist the formation of developed crystals. * cf. Nelson.21A . TERENIN, E . PUTZEIKO A N D I . AKIMOV 87 Fig.3 reproduces the ph-e.m.f. spectrum for both pigments as powders between mica laminae. The sign of the charge carriers in them, obtained by the two methods described above, is positive, either in air, or in vacuo. However, in methyl- chlorophyllide freshly deposited from a chloroform solution a subsidiary maximum at 800 mp appears in the ph-e.m.f. spectrum, and as well in the absorption spectrum of the layer, the charge carriers in which are negative (fig. 3). There is a marked shift of the spectral sensitivity curve for methyl chlorophyllide in the process of formation of larger crystals (fig. 4). This result coincides with that obtained by Jacobs, Holt, Kromhout and Rabinowitch 27 in the absorption mC1 FIG. 4.-Spectral response in oucuo of ph.-e.m.f. in a methyl chlorophyllide (a + 6) layer with microcrystals growing.Curve 1, layer deposited from a (C2H&0 + petr. ether solution ; curve 2, after 10 h contact with H20 vapour at 20°C in 'UQCUO ; curve 3, after a repeated treatment by H20 vapour. The relative heights of the maxima must be 1 : 2 : 3.5 in this sequence. spectrum of ethyl chlorophyllide microcrystals of growing size. For a chloro- phyll layer the spectral maximum of photoelectric sensitivity is already at 720 mp and does not much shift after a similar treatment by H20 vapour (fig. 5). The detachment of an electron from an isolated dye molecule in vacuu requires a photon energy equivalent to 6-7 eV. The threshold of the external photoelectric effect from layers of solid dyes gives for Mg-phthalocyanine 6.3 eV and for ery- throsin 6.5 eV.These values do not significantly change when these dye molecules are adsorbed on ZnO or SnOz. The inner photo-effect observed for crystalline dyes and pigments shows that a photon energy equivalent to 2 eV, or even less, which produces only the excita- tion of a molecular centre of the lattice, is capable of detaching an electron from the molecule. In fact, the active light falls exactly in the range of the absorption band of the dye molecule, modified by the surroundings. The large decrease of the ionization energy of a neutral particle in a solid medium is known for inorganic crystals. It means here that after excitation and probably exciton88 ENERGY TRANSFER I N ORGANIC SYSTEMS migration the molecular electron can pass into a level of lower energy outside the molecule.19 * Since in the pigments studied the carriers of the photo-current are positive holes, this means that the electrons liberated by light are kept for some time in traps, the current being transported mainly by an electron exchange between the molecules at the ground level.l9*20 Our measurements of the relaxation of the photoconduction have established for the phthalocyanines a component of the current with a time constant 5 x 10-5 sec.When the phthalocyanine possesses a central metal atom a slower component with a time constant 5 x 10-3 to I S I.0 c-: E ai r’ I a 0.5 mCL FIG. 5.-Spectral response of ph.-e.m.f. in a chlorophyll a layer in oucuo. Curve 1, before ; curve 2, after contact with H20 vapour for 10 h in vacuo.7 x 10-3 sec is enhanced. Thus the metal atoms seem to create additional attachment levels for the photo-electrons. This is in addition to the slowly decaying component observed during measurements of the d.c. photoconduction. SENSITIZED PHOTO-EFFECT In previously published researches we have shown that the ph-e.m.f. in ZnO, TlHal, AgHal, and in several other n- and p-inorganic semiconductors can be optically sensitized by light absorbed by dyes and pigments of many classes, monomolecularly absorbed on their surface.16-18 This phenomenon has been obtained with the condenser method, and as well with the ordinary transverse photo-conductivity method with constant or intermittent illumination and con- stant potential. The weight of the arguments derived from the various experi- ments, described in the previous papers,6* 149 16-18 favour an energy, and is against an electron transfer, from the excited dye molecule to the inorganic semiconductor.The sharpness of the sensitization spectrum for the chlorophyll and phthalo- cyanine pigments on Zn0,6#14,17 is an argument against any coupling of the * Lyons26 has recently computed the position of such a level for anthracene and naphthalene.A. TERENIN, E . PUTZEIKO AND I . AKIMOV 89 electron in the excited state of the adsorbed dye with the conduction band of the adsorbent. In fig. 6 such a spectrum is given for pheophytine. The striking increase of the sensitized photo-response at + 100" is a phenomenon analogous with the photographic sensitization of AgBr by dyes.It shows that additional thermal energy is required. The peculiar increase of the sensitized photo-response at - 15°C is produced by the increase of the proper ph-e.m.f. of ZnO, on cooling. Microcrystalline chlorophyll powder possesses a very small temperature co- efficient of ph-e.m.f. : at - 190°C the latter is 60 % of the value at 20°C. 4 N % 3 I s! s- E N h 2 G.: E 4 c a I I I i ttoc */ mCL FIG. 6.-Spectral response of the sensitized photo-e.m.f. (reduced to equal incident energy for ZnO with adsorbed pheophytine (a + b) at different temperatures ("C) in vacuo. We have also shown that light absorbed in colloidal aggregates of a dye, viz., of Mg-phthalocyanine or pinacynole, is capable of sensitizing the ph-e.m.f. in ZnO, although less effectively than monomolecularly dispersed dye molecules.6.14 The sign of the charge carriers in Mg-phthalocyanine crystals is positive, whereas ZnO, a typical electronic semiconductor, exhibits always negative carriers in the spectral range of the sensitizing pigment.It is difficult to imagine that a positive hole migrating in the pigment particle would be capable of releasing an electron in ZnO. An exciton migration in the colloidal particle of phthalocyanine from the site of the photon absorption to the surface of ZnO is more plausible. In several combinations of two dyes simultaneously adsorbed on ZnO, we observed a non-additivity of a peculiar kind. Poor photosensitizers, such as safranin T or phenosafranin, which have absorption maxima near 560 mp, adsorbed alone give a slight sensitization maximum at this wave length.However, when one of90 ENERGY TRANSFER I N ORGANIC SYSTEMS these dyes is simultaneously adsorbed on ZnO together with Mg-phthalocyanine or chlorophyll the efficiency of sensitization by light absorbed by these poor sensit- izers is so considerably enhanced, that their maximum predominates in the spectrum (fig. 7). The position of the spectral maxima of both the sensitizers are the same as when they sensitize alone and a close association of both dyes on the surface is unlikely. These experiments seem to indicate that an inefficient sensitizer can pass its excitation energy to a good sensitizer molecule. This intermolecular transfer of energy can evidently be of the same kind as the inductive resonance of the electro- magnetic field type, which is well known for the sensitized fluorescence of a binary solution of dyes and in concentration quenching.4 0 0 SO0 6 0 0 700 800 POD mcL FIG. 7.-Sensitization of the photo-e.m.f. of ZnO by safranin T (560 mp), adsorbed alone (curve l), and by safranin T, adsorbed together with chlorophyll (a + h) (curve 2). The ordinate for curve 1 has a scale 10 times less than that for curve 2. Another alternative would be the assumption that a stepped electron-transfer process is taking place between the poor sensitizer via the good one to the semi- conductor or in the reverse direction. The possibility of an electron exchange as a mechanism of sensitization of the photo-effect in semiconductors has been shown by us not to be plausible.6.14 INTERMOLECULAR PROTON TRANSFER, AT LOW TEMPERATURES, INDUCED BY LIGHT * In a search for a proton transfer between molecules linked by strong hydrogen bonds, which could be induced by light, we have studied in 1947 some binary system of organic acids and bases.7 We then succeeded in observing this effect * by A.Terenin and A. Shablya.A . TERENIN, E. PUTZEIKO AND 1. AKIMOV 91 by using a fluorescent indicator base-acridine, embedded in an organic acid, by joint sublimation in VOCUO on to a surface, cooled by liquid air, The best results have been obtained with oxalic, succinic and terephthalic acids. Such a com- posite layer gave on excitation by 366mp a bright green fluorescence with a spectrum, characteristic of the acridinium cation. After a prolonged illumin- ation (10-30 min) with light in the range of the first, or second maxima of the ab- sorption spectrum of the acridinium cation, the fluorescence became violet, cor- responding to the spectrum of the neutral acridine molecule.By keeping this at low temperature in the dark, the fluorescence spectrum of the acridinium cation was slowly restored.7 The phenomenon could be repeated reversibly several times.* F FIG. 8.-Fluorescence spectra of a 9-oxy-phenylacridine layer at - 190°C in vacuo. Curve 1 , spectrum of the initial form (11) ; curve 2, after 15 min irradiation with the full 1-1.-v. light of the Hg lamp (form I) ; curve 3, restored initial spectrum after staying 90 min at 20°C in the dark. In continuation of this line of research we tried a larger variety of organic acids as a matrix for basic fluorescent indicators, having also in mind amphoteric acidic-basic molecules forming extended arrays, coupled by strong hydrogen bonds. Of the many systems tried the best results have been obtained with 9-oxy-phenylacridine (I), which is known to form, in their crystalline state, long array of molecules tightly bound by acidic-basic hydrogen bonds of the kind A sublimed layer of this compound in vacuo on a surface kept at - 190°C fluoresces with a green colour giving a spectrum characteristic of the ionized form of the molecule 01).This fluorescence is excited by 366 mp through a light filter. However, after a 15-min irradiation with shorter ultra-violet the colour of the fluorescence becomes violet, and the spectrum of the neutral molecule of the * According to the extensive researches of Forster,Z 23 Weller 24 and others in recent years, we now know many instances of a change of the acidic, or basic strength of fluorescent molecules in liquid solutions upon light excitation cf.; also ref.(25).92 ENERGY TRANSFER I N ORGANIC SYSTEMS compound appears. On keeping in the dark for an hour the fluorescence colour reverts to green, i.e. to the spectrum of the ionized molecule and the cycle can be repeated several times without any irreversible change (fig. 8). Fluorescence spectra, similar to those of fig. 8, are observed in an ethanol solu- tion of 9-oxy-phenylacridine at 20°C. In a neutral ethanol solution the fluorescence maximum is situated at 22,300 cm-1 (440 mp). When the solution is acidified by HCl (5 N) the fluorescence maximum is at 20,000 cm-1 (500 mp).The initial spectrum is restored when pyridine (1 N) is added to the solution. Corresponding changes in the absorption spectrum of the solution are observed. The neutral solution possesses an absorption maximum at 360 mp ; in the acidified one an additional absorption maximum at 440 mp appears ; this latter disappears when pyridine is added. The explanation is the same as given before for the binary system, acid + acrid- ine. In that case an intermolecular transfer of proton induced by light from the acridinium cation to the anion takes place, both being linked by strong hydrogen bonds. In the experiments, here described, it is the same bifunctional amphoteric molecule possessing a basic and acid group at both ends which is involved.It is functioning at the same time as a proton donor and a proton acceptor. The inter- molecular proton transfer must be accompanied here by a tautomeric change in the structure of the molecule, and this can be propagated along an array of inter- connected molecules. The condensation of acetic acid on the layer at - 190°C greatly enhances, as an additional medium, the observed reversible shift in the fluorescent colour, evidently facilitating the intermolecular proton transfer. CONCLUSIONS During the years some prominence has been given to the semi-conductivity theory of photosynthesis. The results described above contribute some basic facts which have to be taken into consideration. These are : (i) Solid layers of chlorophyll, the chlorophyllides, pheophytine, the porphy- rines, and as well the phthalocyanines behave like photo-semiconductors, although rather unsensitive ones.(ii) The mobile charge carriers for most of them are electron vacancies, i.e. positive holes. This means that the primarily released electrons are being kept for some time in traps, and the electrons move at the ground level in an inter- molecular oxidation-reduction exchange process. (iii) The excitation spectrum of the photo-conduction, or the photo-e.m.f. is confined in the visible to the absorption band of the molecules forming the lattice. This band is broadened by the close interaction of identical molecules, but remains nevertheless relatively narrow. It seems that we have a system of connected and mutually perturbed, but not fused, electronic clouds of the molecules, i.e.the conduction bands, if they exist, must be quite narrow. (iv) Sensitization of the photoconduction and photo-e.m.f. in inorganic semi- conductors by chlorophyll and the related pigments is an energy transfer to elec- trons trapped at the surface. The described two-component sensitization is very likely due to an energy transfer from a poor sensitizer to a better one. This is reminiscent of the energy transfer from the phycobilines and the carotinoides to chlorophyll. (v) The observed proton transfer induced by light in a layer of acridine deriva- tives, where the molecules are connected by hydrogen bonds, can be regarded as an instance of an acid-base-type of transport at a low energy level.The authors are thankful and greatly obliged to Dr. Yu, N. Sheinker for the loan of many tautomeric acridine derivatives used in this work, and for much helpful advice.A . TERENIN, E. P U T Z E I K O AND I. AKIMOV 93 1 Vartanian, J. Physic. Chem. (U.S.S.R.), 1948, 22, 769. 2 Putzeiko, Compt. rend., U.R.S.S., 1948, 59, 471. 3 Vartanian, Bull. Izv. Acad. Sci., U.S.S.R. (Physic.); 1952, 16, 169; 1956, 20, 1541. 4 Vartanian and Karpovitch, Compt. rend., U.S.S.R., 1956, 111, 561 ; 1957, 113, 1020. 5 Putzeiko and Terenin, J. Physic. Chem. (U.S.S.R.), 1956, 30, 1019. 6 Terenin and Putzeiko, J. Chim. physique, 1958, 55, 681. 7 Terenin and Kariakin, Nature, 1947, 159, 1151 ; Compt. rend. (U.S.S.R.), 1947, 58, 8 Ermolaev and Terenin, J . Chim. physique, 1958, 55, 698. 9 Vartanian and Terenin, J. Physics (U.S.S.R.), 1941, 4, 173. 10 Vartanian, J. Physic. Chem. (U.S.S.R.), 1946, 20, 1065 ; 1950, 24, 1361. 11 Vartanian, Acta physicochim., 1947, 22, 201. 12 Vartanian and Karpovitch, J. Physic. Chem. (U.S.S.R.), 1958, 32, 178, 543. 13 Bergmann, 2. physik, 1932, 33, 209. 14 Terenin, Putzeiko and Akimov, J. Chim. physique, 1957, 54, 716. 15 Tolstoi and Feofilov, Progr. Physic. Sci. (Uspekhi phys. nauk, U.S.S.R.), 1950,41,44 16 Putzeiko and Terenin, J. Physic. Chem. (U.S.S.R.), 1949, 23, 676. 17 Putzeiko and Terenin, Compt. rend., U.S.S.R., 1950, 70, 401 ; 1953, 90, 1005. 18 Akimov, J. Physic. Chem.(U.S.S.R.), 1956, 30, 1007. 19 Terenin, Radiotech. Electron. (U.S.S.R.), 1956, 1, 1127. 20 Eley and Parfitt, Trans. Faraday SOC., 1955, 51, 1529. 21 Nelson, J. Chem. Physics, 1958, 29, 388. 22 Forster, 2. Elektrochem., 1950, 54, 42, 531. 23 Bonitz and Forster, 2. Elektrochem., 1952, 59, 137. 24 Weller, 2. Elektrochem., 1956, 60, 1144; 1957, 61, 956. 25 Kokubun, 2. physik. Chem., 1957, 13, 386 ; 1958, 62, 599. 26 Lyons, J. Chem. SOC., 1957, 5001. 2’ Jacobs, Holt, Kromhout and Rabinowitch, Arch. Biochem. Biophys., 1957, 12, 495. 425.

ISSN:0366-9033    

DOI:10.1039/DF9592700083

出版商:RSC    

年代:1959

数据来源: RSC

摘要:

GENERAL DISCUSSION Prof. H. C. Longuet-Higghs (Cambridge University) said: The work of Prof. Porter, and also that reported by Dr. Bowen in his paper, seem to show that the probability that an electronically excited molecule loses its energy by a non-radiative process is a sensitive function of the viscosity of the medium, and rises sharply as the viscosity falls. If this phenomenon is genuinely unimolecular, rather than due to diffusion-controlled quenching by a molecule such as oxygen, one is forced to the conclusion that the non-radiative energy loss must arise from the purely mechanical motion of the medium relative to the excited molecule. On this hypothesis, one is led to consider the mechanical history of the excited molecule, and to regard the non-radiative energy loss as a transition induced by a fluctuating mechanical perturbation, namely the time-dependent forces exerted on the excited molecule by its neighbours. According to first-order time-dependent theory, the probability per unit time of a transition involving an energy change hv is proportional to the intensity of the corresponding Fourier component of the perturbation.It is therefore the intensity of mechanical '' noise " at frequency v which determines the transition probability. Now there exists a simple connection between the fluctuating forces on a molecule and its diffusion constant D. Briefly, the diffusion constant is deter- mined by the low-frequency components of the fluctuating force, and according to the theory of Brownian diffusion the viscosity is related to the diffusion constant by an equation of the form Dv = 67zakT, where a is the molecular radius.An inverse dependence of radiationless energy loss upon the viscosity 7 therefore indicates a direct dependence of the degradation rate upon D. One is therefore led to the conclusion that it is the low-frequency components of the mechanical noise which are most efficient in removing the energy of the electronically excited molecule. This conclusion depends, naturally, on the reliability of the experimental results. It is, nevertheless, a reasonable one since the non-radiative degradation process is likely to occur in small energy steps. This hypothesis might, indeed, be tested experimentally by examining the effect of sonic or ultrasonic power on the phosphorescence of molecules embedded in highly viscous media.Dr. D. H. Whiffen (Birmingham University) said: One query raised by Prof. Porter is the cause for the much faster rate for the radiationless transition S1-7'1 than for the equally spin-forbidden TI-SO. In the approximation in which the electron wave function can be expressed as the product of one-electron wave functions, the single electron excitation states occur in pairs with the same space wave function, but singlet and triplet spin functions respectively. If S1 and TI form such a pair then the overlap of space wave functions is perfect for S1 and T I , whereas 7'1 and SO are orthogonal in this respect. That is S1-Tl is electronically- allowed, spin-forbidden, whereas T1-5'0 is both electronically- and spin-forbidden and would be much slower.This statement involves drastic assumptions such as the neglect of exchange integrals and configuration interaction, but the problem appears to merit a more sophisticated theoretical treatment. The simi!arity of the space wave functions may also lead to a closer geometry between S1 and T1 than T1 and So, and a more favourable overlap of vibrational wave functions for those degrees of freedom for which the vibrational quantum number is unchanged during the transition. Prof. Longuet-Higgins has given one mechanism for a viscosity dependence of the transition probability though this is equally valid for singlet-singlet and 94GENERAL DISCUSSION 95 singlet-triplet transitions. An additional feature specifk to transitions involving paramagnetic states is the interaction between the rotational motion and the spin- orbit and dipolar spin-spin couplings.These couplings tend to tie the electron spin to the molecular framework whereas the gyroscopic nature of the spins tends to keep these unchanged in direction in space. In mobile liquids, the rotational rate is - 1010 sec-1 and of the same order of magnitude as the spin couplings. The zero-field splitting of the triplet is destroyed and a time-dependent mixing of singlet and triplet states is introduced. This is precisely the feature required to break down the fixed molecule spin-forbidden character so that a large viscosity dependence is reasonable. This would be related to the microscopic rotational viscosity and an exact correlation with macroscopic viscosity is not to be expected.Dr. A. Weller (Stuttgart) (communicated) : Prof. Porter’s interesting explana- tion of the intramolecular triplet state deactivation and of its viscosity dependence involves the following three implications which have not yet been fully proved : (i) The radiationless crossing Sl-T’l in rigid solvents should depend on the ex- citation wavelength in such a way that for excitation within the 0,O-transition this crossing is inhibited and consequently the phosphorescence yield is low, whereas for excitation with light of shorter wavelength the Sl-Tl transition probability increases with increasing excess vibronic energy (which can be used to soften the surrounding medium). (ii) Unusually large Stokes shifts should occur between singlet-triplet absorption and phosphorescence.This seems to be at variance with the results of Evans 1 (cf. table 1 of his paper). (iii) Due to the proposed structural difference between triplet and ground singlet states it is to be expected that difficulties arise with the assignment of vibrational frequences found in phosphorescence spectra to Raman and infra-red frequences. Moreover, analysis of the phosphorescence spectrum and of the fluorescence spectrum should yield quite different results with regard to the vibrational frequences involved. Prof. G. Porter (Shefield University) said : Several interesting suggestions have been put forward as alternatives to our explanation of the viscosity dependence of the rate of radiationless triplet-singlet transitions.Explanations are of two kinds depending on whether energy transfer or the electronic transition is considered to be rate-determining. The original state is a triplet with thermally equilibrated vibrational levels and the final state is the ground level, also in thermal equilibrium. The process must, however, occur in two stages : (i) a change of electronic state which, in the Born-Oppenheimer approximation, occurs isoenergetically and which, in the absence of collisions or radiation, will eventually be reversed : (ii) loss of vibrational energy from the vibrationally excited ground state formed by process (i). Longuet-Higgins’ theory assumes that process (ii) is rate-determining and I think this cannot be correct. The experimental facts are that the lifetime of the triplet state in the gas phase is not dependent on inert-gas pressure and is shorter than the lifetime in solution. But there is a wealth of evidence from vibrational- energy-transfer studies in general that the rate of transfer increases with gas pressure and is very high in solution.The suggestions of Ford and Whiffen, like our own, are based on the assumption that (i) is the rate-determining process. Ford’s ideas are similar to ours in that the requirement is the attainment of vibronic configurations which are “ active ” for radiationless conversion. The principal objection is that a viscosity dependence of rate constant is observed in molecules having little or no symmetry but this does not exclude the possibility that electronic transition probability is still very dependent on nuclear configuration as discussed in our paper.1 Evans, J. Chem. SOC., 1957, 1351.96 GENERAL DISCUSSION The suggestion of Whiffen is a very attractive one. It has been clear to me for some time that the principal effect of solvents in the viscosity range over which the rate constants are affected will be on rotational rate but I was never able to understand how this could affect the electronic transition probability. The time- dependent mixing of singlet and triplet states produced by interaction between rotational motion and spin-orbit coupling is just the kind of effect which is required and this suggestion merits further study, both experimental and theoretical. Whether it will eventually be necessary to retain the postulate of a “bent” triplet state in molecules like benzene remains to be seen and Dr.Weller has mentioned several points which need further investigation in this connection. On the other hand, there is little doubt that distortion of the molecule in the excited state is important in increasing radiationless transition probabilities in some molecules as is shown, for example, by the rather generally observed decrease of fluorescence yield when planarity of a molecule is no longer enforced. Prof. R. Livingston (University of Minnesota) said: Dr. Ashley Pugh and I have measured the rate of decay of the anthracene triplet in a series of hydrocarbon solvents, ranging from pentane to decalin, and essentially confirm the findings of Dr. Porter and Dr. Wright. As was reported earlier for chlorophyll solutions, the disappearance of the triplet state of these molecules is due to concurrent first- and second-order reactions. The second-order process is strongly dependent upon the viscosity of the solvent and at least for the more viscous solvents, appears to be diffusion-limited. How- ever, for solvents of low viscosity, our values for the second-order rate constants are about half as great as those of Porter and Wright.This discrepancy is much larger than the random variation and must be due to some systematic error, possibly in the estimation of the effective extinction coefficients of the triplet state for the light used for the photometric analysis of the solutions. It is relatively difficult to obtain reproducible values for the first-order rate constants.We believe that this variability is caused by the presence of quenching impurities in the solvents. We were usually able to obtain minimal values for the rate constants by storing the (hydrocarbon) solvents over sodium and dis- tilling off a sample immediately before it was to be used. It is probable that the very high value which Porter and Wright obtained for tetrahydrofurane is in- dicative of the presence of an impurity in this solvent. Our results indicate that, except for very viscous solvents like glycerol, the effect of viscosity upon the first- order rate constant is small. Prof. G. Porter and Dr. M. R. Wright (Shefield University) said : The derivation of accurate first- and second-order constants from a composite reaction presents considerable difficulty and it is essential to obtain measurements over a wide con- centration range.Values of the first-order rate constants are also very sensitive to impurities, particularly oxygen. Nevertheless, our work in hexane, water and ethylene glycol gave very satisfactory reproducibility over several years and in different solvent samples. The results in viscous paraffins were also quite repro- ducible but were somewhat dependent on the solvent sample as might be expected. The value for anthracene in tetrahydrofurane was obtained from one solvent sample only and may therefore be high. It is difficult to question the statement that “ except for very viscous solvents like glycerol, the effect of viscosity upon the first-order rate constant is small ” but our work shows, for example, that the constant decreases by a factor of more than ten in passing from hexane to glycol as the viscosity increases seventy times.After many attempts to explain viscosity effects entirely in terms of bimolecular processes we are now satisfied that the viscosity dependence of the first-order triplet decay has been established. Mr. F. Wilkinson (Shefield University) said: Porter and Wright have dis- cussed in their paper the quenching of the triplet state by paramagnetic ions. Another quenching process which does not violate Wigner’s rule is A* (triplet) + Q (singlet) -+ A (singlet) + Q* (triplet).GENERAL DISCUSSION 97 The necessary conditions for unambiguous transfer of this type are that the quencher should have a triplet level lower than that of A and also that its first excited singlet level should be higher than that of A, to exclude any transfer between singlet levels.Terenin and Ermolaev 1 have studied transfer from the triplet states of benzophenone and a number of similar compounds to naphthalene and its derivatives. This was in rigid solvents and the quenching-rate constants ob- tained were therefore rather low. Prof. Porter and I have studied these and other systems where both the donor and the acceptor molecules are aromatic hydrocarbons, e.g. triphenylene and naphthalene. Using the method of flash photolysis, we have been able to observe energy transfer in ordinary solutions at room temperature. Under appropriate conditions, using a soda-glass reaction cell as filter, transient spectra can be re- corded from benzophenone and from triphenylene with benzene as solvent. Naphthalene alone shows no transient as no light is absorbed.However, upon mixing either benzophenone and naphthalene or triphenylene and naphthalene, keeping the concentrations identical with those above, the well-known triplet absorption spectrum of naphthalene appears, but absorption from the transients of the donors, benzophenone and triphenylene, are not observed. So far only a lower limit of the quenching rate constants can be given. These are 108 1. mole-1 sec-1 for naphthalene and benzophenone and 109 1. mole-1 sec-1 for naphthalene and triphenylene. We intend to obtain these quenching rate constants in the near future. These may well come very close to the calculated value of 1010 1.mole-1 sec-1 for a bimolecular reaction in benzene solution at 20°C determined entirely by the rate of diffusion of the reactants towards each other. In view of the high rate constant and long triplet-state lifetime, this type of energy transfer may be of very great importance in solution, even compared with singlet-singlet transfer. Prof. A. Terenin (Leningrad) said: I cannot agree with the statement originally made in the paper of Prof. Porter and Dr. Wright, that the rate constants for the energy transfer triplet D -+ triplet A, observed by Ermolaev and myself in rigid solvents should be very low and that they are not yet known in ordinary solutions. This latter case has been recently studied by Backstrom and Sandros2 in the quenching of the biacetyl phosphorescence in a benzene solution at 20°C.On addition of polynuclear aromatic hydrocarbons with a suitable position of the triplet level, the biacetyl phosphorescence was strongly quenched. The transfer efficiency has been found of the same magnitude as for the quenching of oxygen, i.e. of the order of 1010 1. mole-1 sec-1. The remarkable results just presented in the discussion from the laboratory of Prof. Porter confirm this conclusion. In this connection I would like to present our estimation of the energy transfer between the triplet levels as observed in the sensitized phosphorescence of rigid solutions. We have previously shown 3 that this transfer occurs at close distance and that its probability is definitely larger than the probability of emission from the triplet level of the donor, which lies in the range 103-10-1 sec-1.Moreover, we have shown that the phosphorescence quenching of the energy donor by the acceptor closely obeys the formula IO/& = exp ac, where 10 is the initial phor- phorescence intensity of the donor, and Ic that when the acceptor concentration is c (a being a constant). The same expression has been deduced a long time ago by F. Perrin for the self-quenching of fluorescence by concentration increase. His assumption was that there is a sharply delimited sphere of action around the donor such that when an acceptor molecule happens to enter it, there would occur an instantaneous quenching. It has been found, however, that the concentration quenching of fluorescence does not obey this exponential law at all. On the 1 Terenin and Ermolaev, Trans.Favaday Soc., 1956, 52, 1042. 2 Backstrom and Sandros, Acta Chem. Scand., 1958,12, 820. 3 Ermolaev and Terenin, J. Chim. physique, 1958, 55, 698. D98 GENERAL DISCUSSION contrary, the assumption of Perrin is realized in our case of triplet -> triplet energy transfer. From the formula given, a value of 12-13A is obtained for the radius of the sphere of instantaneous energy transfer from the triplet phosphorescent molecule to another one, transferring it to the triplet state. At the closer distance in organic lattices, the same type of energy transfer could then take place in some cases even more efficiently than the singlet 4 singlet transfer. Indeed, in concentrated dye solutions there is in addition to the known inductive transfer of singlet excitation energy, also a dissipation of energy.A strong concentration fluorescence quenching occurs already at c1/2 = 10-2 mole 1.-1. This corresponds to a mean intermolecular distance of ca. 30A. At closer ap- proach of 16 A (c = 0.1 mole 1.-1) the intensity must decrease to 1/20 of the initial value, and still more for the intermolecular distances in a solid. It may therefore be presumed that if the excited molecule has passed from the singlet to the triplet level, the energy of this state can be handed over and propagated along the array of closely packed molecules as efficiently as by the exciton mechanism valid for the singlet state. This means that a " triplet exciton " must be also taken into account.Prof. G. Porter (Shefield University) said : The rate constants determined by Ermolaev and Terenin for energy transfer from the triplet state in rigid solvents were in the range 3 x 10-1 to 5 x 1021. mole-1 sec-1 which are very low com- pared with quenching constants in solution discussed in our paper. This is merely a statement of fact and it is in no way surprising since diffusion is prevented. The data of Backstrom and Sandros (which were not available when our paper was submitted) and also the data presented by Wilkinson at this meeting now show that rate constants of some similar processes in fluid solvents are very high and may approach the maximum rate of a diffusion-controlled reaction. Clearly energy transfer of the type triplet D -+ triplet A is one which must be considered quite probable both in fluid solvents and in rigid media.Dr. J. P. Simons (Birmingham University) said : With reference to the paper of Prof. Porter and Dr. Wright, I should like to make some observations on the conservation of overall spin angular momentum in systems where chemical, as well as physical, processes may occur. For anthracene, for example, self-quenching of the fluorescence can lead to dimerization. This has been shown to occur exclusively through the singlet state, since investigations by Prof. Porter and by Prof. Livingston have failed to detect any self-quenching of triplet anthracene by unexcited anthracene. This is understandable if overall spin must be conserved. The same considerations apply to many other polynuclear aromatic hydrocarbons.The observed self- quenching of triplet chlorophyll may occur because of the presence of the magnesium atom. I have recently made some preliminary investigations into the reaction between excited anthracene and maleic anhydride in dioxane. Since the fluorescence of the anthracene is quenched, the excited anthracene must be in the singlet state. Accordingly, this reaction could be regarded as a general case of photochemical addition of which the dimerization of anthracene is a special case. Prof. Norrish and I have also been able to measure the rate constant for the quenching of triplet anthracene by the polystyryl radical, together with the activation energy.' The quenching is of a chemical nature, the triplet anthracene adding to the doublet polystyryl radical : 3A + 2-- M* -+ 2 - MA.The reaction has a rate constant - 1011 1. mole-1 sec-1 and a zero energy of activation, and may be compared with the quenching of triplet anthracene by nitric oxide. 1 Norrish and Simons, Pruc. Roy. Suc. A , 1959,251,4.GENERAL DISCUSSION 99 Prof. J. Weiss (University of Durham) said: In our work on the quenching of fluorescence of various polycyclic hydrocarbons, such as anthracene, 3 : 4- benzpyrene, 20-methylcholanthrene, etc., by oxygen and nitric oxide,lab we found that the rate constants of fluorescence quenching were of the order of k - 1010 to 1011 1. mole-1 sec-1, i.e. very close to the values reported by Porter and Wright for the reactions with the triplet state. We also investigated the action of various paramagnetic ions such as Mn2f and Er3f but found no quench- ing effect on the fluorescence, from which we concluded that quenching by 0 2 and NO was not connected with their paramagnetism.lb The explanation which we put forward was, that fluorescence quenching is due to an electron transfer, as was established previously in many other cases of fluorescence quenching.lb In the particular case of fluorescence quenching by 0 2 and NO, where the quenching effect is reversible, we suggested a reversible electron transfer, which from a physical point of view, would correspond to an electron exchange.It can easily be shown that such a process would be fully compatible with spin conservation, e.g.: (A*, excited singlet molecule). A+(t).02(? J.? > + A ( ? J.)+O2(? f). (1b) Basically, therefore, the mechanism consists of an electron exchange through the intermediate formation of an ionic complex. Porter and Wright’s suggestion, no the other hand, wouId mean an electron exchange (spin exchange) based on the intermediate formation of a homopolar complex. Apart from purely chemical grounds, the formation of an ionic complex seems more probable since one is dealing here with the long-range Coulombic potential (CC r-1) whereas the homo- polar interaction potential has only a very short range and falls off very rapidly, viz. cc exp (- ar). It seems reasonable to suggest that the triplet state is geometrically different from the singlet state. However, it is difficult to understand that this alone should be the explanation for the slow conversion of the triplet state to the ground state, in a “ frozen ” matrix, because the transition from the first excited singlet state to the triplet state should then be likewise inhibited for similar reasons, whereas, in fact, this latter process is a very fast one with a rate constant greater than 108 sec-1, as pointed out also by Porter and Wright.Therefore, the main reason for the slow radiationless conversion of the triplet state in a frozen matrix must be a different one. I think that one should take into account that these transitions are processes in which electronic excitation energy is transformed into kinetic energy and that one is dealing here with non-adiabatic processes. From the theory of non-adiabatic reactions, as given particularly by Landau and others 2 it follows that the transition probability w is given by an expression of the form : where U is the energy difference at the “ crossing point ” of the potential curves which is related to the energy difference at infinity (A) by the equation : U = A + Wi- Wh (3) where Wi and Wf are the interaction energies in the initial and final state respec- tively and v is the relative velocity, everything taken at the ‘‘ crossing point ”.1 f i - Ff I stands for the angle between the potential curves at the “ crossing point ”. 10 Weil-Malherbe and Weiss, Nature, 1942, 149,471 ; J. Chem. SOC., 1944, 541. Ib Weiss, Symp. SOC. Expt. Biology, 1951, 5, 141. 2 cf. Massey and Burhop, Electronic and Ionic Impact Phenomena (Oxford, 1952).100 GENERAL DISCUSSION Without specifying any particular process, this expression should hold both for an intramolecular as well as for an intermolecular radiationless conversion.In going from the triplet state to the ground state, the energy difference U is com- paratively large and, in a solid matrix, the relative velocity v either within the molecule or of the molecular motion relative to its surroundings is expected to be very small. This will be particularly true here as the triplet state is formed from the excited singlet state by a non-radiative process and should, therefore, be in its lowest vibrational levels. Thus the transition probability to the ground state, according to eqn. (2), may be very small. The situation, however, could well be rather different for the transition from the excited singlet to the triplet state because the excited singlet state is created by an optical absorption and, in general, should be in a vibrationally excited state. Moreover the energy difference between the first excited singlet state and the triplet state is frequently much smaller.All this would, therefore, combine to give an increased transition probability as the energy difference is smaller and v much larger than for the former process. For the same reason, it is quite understandable that the lifetime of the triplet state in the gaseous phase is much shorter than in a solid matrix because in the gaseous phase one is generally working at higher temperatures where some of the vibrational degrees of freedom are likely to be excited.This will lead to an increased relative velocity intramolecularly and also with regard to the other molecules which are in collision with the excited molecule, and hence to an in- creased transition probability. Prof. G. Porter (Shefield University) said : The eqn. (la) proposed by Weiss can certainly lead to spin change of A with overall spin conservation although to show this diagramatically would require a fuller vector diagram. In fact, Weiss’s example is a special case of our theory in which he has discussed the nature of the complex in more detail. There is nothing in our theory which excludes charge- transfer complexes and indeed we think this will be a very common type. But we should not want to limit the theory to any specific type of interaction since all that is necessary is that the triplet and the paramagnetic catalyst shouldap- proach to a distance at which spin-spin interaction (or exchange) becomes sig- nificant.It is not even necessary that any stable complex be formed at all, although increasing the lifetime of the complex will clearly increase the probability of spin exchange. Dr. Mason has asked whether the effect of paramagnetic ions may not, after all, be a true magnetic perturbation but one in which the correlation with magnetic susceptibility is obscured by solvent effects and complexing of the ion. Clear evidence that this is not the case comes from comparison with the very similar process of nuclear spin conversion in ortho-para hydrogen, which can actually be used for the measurement of magnetic susceptibilities of ions in water solution.We have also used water solutions and the comparison is therefore exact and shows that, if magnetic perturbation of spin-orbit interaction were the factor responsible a direct relation with magnetic susceptibility would be found under our conditions. I do not understand Prof. Weiss’s point about the triplet state being formed in its lowest vibrational levels. On the contrary, it is formed by crossing from a higher electronic state and will therefore be originally in a high vibrational level whilst the upper singlet can lead to triplet even when the excitation wavelength is very close to the zero point transition. The explanation of differences in triplet lifetimes in gaseous and rigid media in terms of temperature cannot be the general one since very long triplet lifetimes may often be observed in room-temperature glasses.Dr. R. A. Ford (Mullard Research Laboratories, Salfords, Surrey) (cornniuni- cated: A possible explanation of some of the temperature effects on fluorescence observed by Bowen can be found on symmetry grounds for anthracene and its derivatives, With anthracene (point group Vh) the fluorescent state is soGENERAL DISCUSSION 101 that radiationless conversion 1B2U-+1Alg is forbidden between the zero-vibra- tional level of lBzu and all totally symmetric vibrational levels of the ground state (Kronig's rules).l state could yield vibronic configurations capable of combining with appropriate vibronic configurations of the ground state. The efficiency of the conversion would then depend on the matrix elements.(i) I Hp 1 l A l g ( j ) ) between the ith and jth vibronic states, Hp being the appropriate perturbing operator. The temperature coefficient of fluorescence should then be related to the energy of the " active " vibronic configuration of This will also be the case with symmetrically substituted anthracenes, such as 9-, lo-, but unsymmetrical sub- stitution destroys the centre of symmetry and reduces the point group to C,. Then the gt-tg, u++u, g t / - + u selection rule for radiationless transitions no longer holds and conversion " A -+'' A "I may occur 2 from the zero-vibrational level of " A "II. We may therefore expect a much reduced temperature coefficient for these derivatives. However, that this is not the whole story is clear from the fact that Bowen has found anthracene to have a middle-sized temperature coefficient in between the 9-, 10- and side-substituted molecules.Also 9 substitution, which reduces the point group to CzV results in an increase in temperature coefficient. Clearly there is something special about substitution at the 9 and 10 positions which is independent of symmetry. The possibility that changes of shape between the l A l g , and 3BzU states are involved cannot be ruled out, but inspection of the vibrational structure in absorption,3 fluorescence 3 and phosphorescence 4 suggests that they are not very important since the (0,O) bands are prominent in all these spectra. Similar rules will hold for radiationless transitions from the triplet state except that here the conversion will be controlled by the symmetry properties of the inter- mediate singlet state which perturbs the triplet, not by the triplet itself.Since the spin-orbit coupling operator transforms under Vh as B1,(,), B2g(y) and B3g(x) the intermediate states which will mix with the lowest triplet state of anthracene, presumably 3B2*, are It is not possible to decide which of these states is the important intermediate state without reference to the polar- ization of phosphorescence but we may note that it must be an " odd '' electronic state. Therefore, radiationless conversion to totally symmetric vibrational levels of the ground state will require the excitation of " odd " vibrational quanta of the intermediate singlet state.If the effect of viscosity is to modify the vibra- tional energy distribution in the solute molecule by damping certain modes of nuclear motion which may contribute to vibronic configurations which are " active " for radiationless conversion, the dependence of triplet state lifetime on viscosity observed by Porter et al. may be explained in a similar way. It would be very interesting if unsymmetrically substituted anthracene derivatives showed a much smaller viscosity dependence of triplet state decay than anthracene itself. Prof. H. Kallmann (New York University) (partly communicated) : The experi- ments of Bowen on the quenching of the singlet state and those of Porter et al. on that of the triplet state show very clearly the influence of solvent viscosily upon quenching rate.It must be, however, realized that the influence of viscosity and temperature upon light emission actually are not always so clearly established. Investigations in our laboratory have shown that the emission of certain molecules with large quenching constants is indeed increased if a solvent of higher viscosity is used (e.g. paraffin oil). But no such increase results in other solvents (e.g. silicon oil), though it has a viscosity comparable to that of paraffin oil; and in However, excitation of " odd " vibrational quanta of the 1AlU, 1Blu.5 1 Kronig, 2. Physik, 1928, 50, 347. 2 Roman subscripts I, 11, . . ., define different electronic states of similar symmetry. 3 Sidman, J. Chem. Physics, 1956, 15, 115. 4 Padhye, McGlynn and Kasha, J.Chem. Physics, 1956,24, 588. 5 McClure, J. Chem. Physics, 1949, 17, 665.102 GENERAL DISCUSSION plastics, in spite of their rigidity, little increase of light emission is generally observed. This indicates that the external viscosity does not describe completely the internal influence on the emitting molecule. On the other hand, there are also molecules whose light emission is practically not at all increased by an increase in vjscosity. Bowen points out that there may be two different processes for quenching, one depending on T and another, less dependent on these properties, which may be connected with the transition from the excited singlet to the triplet state. In- vestigations by Dr. A. Adelman of this laboratory seem to show that this latter transition is also influenced by the surrounding medium.Pure naphthalene crystals display no phosphorescence at low temperature either under light or under high energy excitation. But the addition of impurities such as benzo- phenone to naphthalene makes phosphorescence appear with a well-defined lifetime of 0.4 sec which means a quenching to about 20 %. The excitation occurs via the singlet state of naphthalene, as is concluded from excitation by light of X = 313 mp and by high energy. The addition of an impurity changes either the transition probability triplet-ground state or that of the excited singlet- triplet state or both. The investigation seems to indicate that it is the latter transition which is forbidden in pure naphthalene crystals and increased by impurities.In durene crystals, phosphorescence occurs in the pure crystal. If naphthalene is added, naphthalene phosphorescence occurs (again excited via the singlet state) with almost unquenched lifetime at 93°K and with a lifetime of 0.1 sec at room temperature. In this system, the naphthalene phosphorescence occurs even at room temperature quenched by a factor of only 5 and also reduced respectively in lifetime compared to the value at low temperature. Such a quenching indicates an activation energy of less than 0.01 eV for the process. Prof. G. Porter (Shefield University) said : I agree with Prof. Kallmann that viscosity alone is not a good measure of the effects which we describe. Windsor and I 1 also examined silicone oils and found, like Kallmann, that at high vis- cosities they were much less effective than a paraffin of similar viscosity.In an extreme case like gelatin the rate constants were not very different from those of water. Clearly it is the microscopic viscosity seen by the excited molecule which is important and diffusion coefficients, if they were known, would probably be a better indication of this parameter. Prof. D. Eley (Nottingharn University) (partly communicated) : Dr. Kallmann has referred to the effect of dissolving fluorescence solutes in rigid polystyrene. Speaking in the broadest sense, the motion of a complete polymer molecule results from the co-operative motion of its constituent segments, according to the well- known model of Kauzmann and Eyring.2 The loss of fluidity of a polymer melt is associated with the formation of a network of molecules, which prevents a translation of whole molecules, although segmental translation and rotation is still active.It seems likely that on further lowering the temperature through the second-order transition point 3 (glass-transition), there is a loss of segmental rotation and I should like to ask Dr. Kallmann if he has observed the quenching of fluorescence for a dye in a polymer as the polymer is passed through the second- order transition point. I gather from Prof. Dole that the nature of the glass-transition is still obscure. Perhaps I could invert my suggestion, to using fluorescence of solutes as an aid to investigating the nature of the glass-transition. A correlation of fluorescent solute behaviour, with the relaxation times of the plastic determined by mechanical and electrical loss measurements might prove interesting.Dr. B. Rosenberg (New York University) said: In considering the two possible quenching mechanisms for the excited substituted anthracene molecules, the direct 1 Porter and Windsor, Proc. Roy. SOC. A , 1958, 245, 238. 2 Kauzmann and Eyring, J. Amer. Chem. SOC., 1940,62, 31 13. 3 Boyer and Spencer, Adv. Colloid Sci., 1946, 2, 1.GENERAL DISCUSSION 103 quenching to the ground state and the quenching by internal conversion to the triplet state, Prof. Bowen has assigned the first as the temperature-dependent effect and the second as the temperature-independent effect. I should like to present some evidence from photoconductivity work which indicates perhaps that the assignments ought to be reversed.When crystals of organic materials such as anthracene are irradiated with light which populates the first excited singlet state, photoconduction occurs. The photocurrent increases with temperature according to the form : i(T) = io exp (- E/kT). It has been found that in all known cases, the activation energy E correlates very well with the vibrational energy of the excited state. For anthracene, E = 0.17 eV. The interpretation of this fact (taken in conjunction with other facts) is that the photocurrent arises mainly from molecules in the triplet state and that in these cases one or two vibrational quanta are necessary for a large transition moment for intersystem crossing.1 This leads to the conclusion that the fluorescence efficiency of the crystal should follow the law given by Dr.Bowen if one assumes no direct quenching to the ground state : 1/F = 1 + K exp (- E/kT), where E should be equal to 0.17eV for anthracene. An attempt was made to determine this. The above law was followed, but the value of E was 0.14 eV. While the above interpretation is still controversial, the evidence seems fairly strong that photoconduction occurs after the molecule has left the excited singlet state in a temperature-dependent process. It is extremely difficult to see how direct quenching to the ground state can lead to photoconduction and therefore strongly suggests that the temperature-dependent process should be associated with the transition to the triplet state. Mr. G. Jackson (Shefield University) said: Referring to the work of Prof, Forster and Dr.Weller on the acid dissociation of the excited singlet state, Prof. Porter and I have been doing work on similar lines concerning the triplet state. Whereas it was found that for aromatic acids and bases in aqueous solution the equilibrium constant of dissociation of the first excited singlet state varies con- siderably from that of the ground state, in the triplet state it appears that no such large difference occurs. Some preliminary measurements are tabulated below, together with the results of Forster and Weller for comparison. The triplet pKs are the mean of measurements by two independent methods, (i) flash photo- lysis of aqueous solutions, and (ii) from phosphorescence data. molecule PK ground state first singlet first triplet 15-naphthol 9.6 3.1 8.3 8-napht hylamine 4.1 -2 3.5 8-naphthoic acid 4.2 10-12 4.2 a-naphthoic acid 3.7 10-12 4.6 acridine 5.5 10.7 5.5 Apparently triplet excitation in these molecules does not seriously affect the electronic configuration at the point of proton attachment suggesting that the triplet is essentially of 7r-n character and the singlet essentially n - n .Prof. H. Kallmann (New York University) (partly communicated) : The experi- ments of Stevens on the lifetimes of benzene and anthracene in the vapour phase pose interesting questions about the comparative behaviour of these substances as gases and as solutes. The measured lifetime of anthracene in solution is 4 x 10-9 sec (molecular fluorescence) which gives an emission lifetime T~ of about 15 x 10-9 1 Rosenberg, J.Chem. Physics, 1958, 29, 1108.104 GENERAL DISCUSSION sec (lifetime without quenching); for benzene the respective values are 9 x 10-9 sec and 300 x 10-9 sec.1 Results 2 have also been obtained for diphenylanthra- cene; r e is around 15 X lO-9sec. These figures are also in rough agreement with those calculated from absorption measurements. Assuming that this is generally the case, consider the of p-terphenyl which is about 2 x 10-9 sec. Since its total absorption is about 5 times that of anthracene, one would expect a life- time for anthracene larger than 10-8 sec, and for benzene larger than 100 x 10-9 sec. The question arises why the time constant r e in the gaseous state of anthra- cene is considerably shorter than the 7, in solution. (For benzene the values for gas and solutian seem to be in agreement.) I was informed that values for anthracene in gaseous state similar to those of Stevens were also obtained by direct measurement by other investigators.One may conjecture that the lifetime measured in solution is increased by re-absorption, but the respective measure- ments of Knau were made at such small concentration that this possibility is excluded. Dr. B. Stevens (Shefield University) (communicated): In reply to Prof. Kallmann’s question, I would like to point out that lifetimes estimated from quenching measurements depend on the square of the value assumed for the collisional quenching diameter. However, if we accept the value 1.0 f 0.2 for the quantum yield of an isolated anthracene molecule,3 the experimentally measured lifetime of 2-8 x 10-9 sec gives 2.8-3.5 x 10-9 sec for the emission lifetime which agrees well with the extrapolated value of 4.3 & 0.5 x 10-9 sec measured directly for the vapour using the phase-shift technique.4 I think the real question here concerns the much longer emission lifetime of anthracene calculated from measure- ments made in solution.It should be remembered that the effects of re-absorption are determined by the optical density of the medium between the emitting molecule and the detector and not by concentration alone; thus even at very low con- centrations the measured lifetime may be appreciably lengthened by radiation imprisonment if a long absorption path is used to obtain a signal of the required intensity.In this respect it is interesting to note that the directly measured life- time of anthracene vapour at a concentration of 10-5 mole/l. is already some 50 % longer than the extrapolated value at zero concentration.4 Dr. G. Weber (Shefield University) (communicated) : The equation r = qr,, where 7, is the emissive or natural lifetime, r the measured lifetime and q the quantum yield applies only if the radiationless transition processes that decrease the quantum yield are competitive with the emission. For long-lived ground state complexes formed between a fluorescence molecule and a quencher molecule Sy6 q is known to vary independently of r. The change in yield in going from the vapour to the solution may be a similar case in which differences in ground-state relations between solvent and solute molecules are responsible for the change in q without parallel change in r.Dr. B. Stevens, Mr. P. J. MeCartin and Mr. E. Hutton (Shefield University) said : We have recently photographed the slow (10-3 sec) component of phen- anthrene-vapour fluorescence, and find that the spectrum contains bands which coincide in position with those observed in anthracene-vapour fluorescence, but which have different relative peak intensities. These bands are of the same in- tensity as that of the slow phenanthrene emission obtained on the same plate but are not observed in the total (fast and slow) emission from the same cell using much shorter exposures. Absorption measurements in solution showed that the phenanthrene contained one part in 104 of anthracene which would provide a 1 Knau, 2.Naturforschung, 1957, 12a, 881. 2 Brucker and Kallmann, Physic. Rev., 1957, 108, 1122. 3 Stevens, Trans. Faraday Soc., 1955,51, 610. 4 Hardtl and Scharmann, 2. Naturforsch., 1957, 12a, 715. 5 Weber, Trans. Faraday SOC., 1948,44, 185. 6 Epple and Forster, 2. Elektrochern., 1954, 558, 783.GENERAL DISCUSSION 105 pressure of anthracene vapour of ca. 10-3 mm under the experimental conditions. We are led to the tentative conclusion therefore that the long-lived (ca. 10-3 sec) excited collision complexes of phenanthrene which are responsible for the slow component 1 also sensitize the fluorescence of the anthracene present ; the " sensitizing half-pressure " of anthracene required would be in the region 10-3- 10-4 mm.A re-investigation of the self-quenching of anthracene vapour fluorescence has confirmed the recent observation2 that this has a negative temperature co- efficient. On the grounds that this is due to the increased dissociation of the excited collision complex to produce the original excited monomer at higher tem- peratures, our results indicate that the excited collision complex has an energy of some 3-6 kcal below the excited singlet monomer. It is generally recognized that the role of transferring species is played by either the excited singlet state of a molecule or by the lowest triplet state which has a longer lifetime but a lower energy. In view of our preliminary findings we would like to suggest a third transferring species, namely the excited dimer or collision complex which has virtually the energy of a singlet state and the lifetime of a triplet.If such a comparatively long-lived complex can be formed by saturated hydrocarbons then perhaps the efficiency of radiation protection by energy transfer in such systems could be better understood. Dr. J. B. Birks (Manchester University) said: I wish to discuss energy transfer in polystyrene solutions. It has been shown clearly, by three independent in- vestigations,2-5 that radiative transfer occurs and accounts for at least 20 % of the energy transfer observed in these systems. Some of the difficulties experienced by Brown, Furst and Kallmann 6 in interpreting their results are due to neglect of this radiative component.For example, the steep initial rise in the intensity- concentration curve of polystyrene + fluoranthene solutions (fig. 7 6) is similar to that of polystyrene + TPB solutions,3~4 and is probably due to radiative transfer. Brown et aZ.6 distinguish between solutes like PPO, where the intensity reaches a maximum at c - 0.05 M, and fluoranthene and chrysene, where the intensity continues to increase beyond c = 0.1 M. This effect has been previously studied in detail4 for a wide range of solutes including PPO. The magnitude of the non-radiative transfer parameter a, which determines the rate of rise of the intensity-concentration curve, is found to be approximately proportional to (.lo#, where is the Forster transfer probability integral, determined by the overlap of the solvent emission spectrum fo(v) and the solute absorption spectrum E~(v).(.lo# is pro- portional to V, the volume of the transfer sphere. u increases4 from zero for biphenyl (negligible spectral overlap), through diphenylbutadiene, naphthalene and anthracene (intermediate overlap) to p-terphenyl, quaterphenyl, PPO and TPB (large overlap). It is clear from the results of Brown et aZ.6 that fluoranthene and chrysene are solutes with intermediate spectral overlap with polystyrene. In considering non-radiative transfer, the main interest is in deciding whether this occurs primarily by the migration process or by the single-step process. In any particular rigid system this will be determined by the relative magnitudes of 1 Williams, J. Chem. Physics, 1958, 28, 577.2 Hardtl and Scharmann, Z. Naturforsch., 1957, 124 715. 3 Birks and Kuchela, this Discussion. 4 Swank and Buck, Physic. Rev., 1953, 91, 927. 5 Krenz, Trans. Faraday Soc., 1955, 51, 172. 6 Brown, Furst and Kallmann, this Discussion.106 GENERAL DISCUSSION tol, the solvent-solute transfer integral, and JOO, the corresponding solvent-solvent Jransfer integral. Unfortunately the usual equations for the migration and single- step processes and (3) yield similar fNR against c curves, and it is difficult to distinguish between them from intensity-concentration measurements. Thus our results 1 on polystyrene + TPB solutions, which agree within the experimental error with (2), are equally consistent with (3), taking 1/ V = 8 x 10-3 M,fmax = 0.5, corresponding to a radius of the transfer sphere of 37 A.The alternative non-radiative processes may be distinguishable by the form of the fluorescence decay of the solute emission.2 In the migration or diffusion process the decay is exponential ; in the single-step process, the decay becomes slower than exponential. Accurate measurements have been made by Swank et aE.3 of the scintillation decay of solutions of p-terhdenyl in toluene and in polystyrene, and the nature of their results is confirmed by Burton and Dreeskamp.4 In the liquid solutions, where the transfer is by diffusion and/or migration, the decay is ex- ponential. In the polystyrene solutions, the decay deviates strikingly from the exponential, in the manner predicted for the single-step process. It might therefore be concluded that in systems with a high Jol, transfer occurs primarily by a single- step process.Note Added in Proof.-Such definite conclusions in favour of single-step transfer are, however, premature since there are at least two other mechanismb which can lead to a non-exponential scintillation decay. Harrison,s Brooks 6 and Owen 7 have observed slow decay components in many organic scintillators, and it has been proposed 8 that these originate from higher triplet states populated by ion recombination, thus accounting for the dependence of the relative magnitudes of the fast and slow components on the ionization density. The decay time of the first slow component is generally 3-4 x 10-7 sec, which is a factor of 10-30 times that of the " tails " of the decay curves observed by Burton and Dreeskamp,4 though an extension of these to lower intensities might remove this discrepancy factor.Their present measurements are, however, more consistent with an alternative mechanism, namely that the slow decay is due to the radiative transfer com- ponent, whose magnitude is - 0-1 of the fast non-radiative transfer component,l~ 9 and whose decay time is increased by that of the polystyrene fluorescence 3 which is - 1-5 x 10-8 sec. This explanation is preferred, since it agrees well with the existing experimental data.3.4 It has been shown elsewhere 8 that the behaviour of polystyrene + TPB solutions can thus be quantitatively described in terms of non-radiative migration and transfer and radiative transfer processes. Comparison of our results 1 with (3) indicate that for polystyrene + TPB solu- tions fNR tends to f m a = 0.5 at high c.A similar value for fma is obtained by comparison with the scintillation data 9 (fig. 4 1) and extrapolation to high c. Similar results withf,,, = 0.5 have been obtained for toluene + terphenyl solu- tions.10 It is concluded that, even with the best solvents, the transfer from the 1 Birks and Kuchela, this Discussion. 2 Galanin, J. Expt. Theor. Physics, U.S.S.R., 1955, 28, 485. 3 Swank, Phillips, Buck and Basile, Z.R.E. Trans. Nucl. Sci., 1958, NS-5, 183. 4 Burton and Dreeskamp, this Discussion. 5 Harrison, Nucleonics, 1954, 12 (3), 24. 6 Brooks, Nuci. Instr, Methods, 1959, 4, 151. 7 Owen, I.R.E. Trans. Nucl. Sci., 1958 (N.S.) 5, 198. * Birks, Conference on Dielectric Devices, Birmingham University, Sept.1959, Proc. Physic. SOC., to be published. Swank and Buck, Physic. Rev., 91, 917. 10 Birks and Cameron, Proc. Physic. Suc., 1958,72, 53.GENERAL DISCUSSION 107 first excited singlet state is not 100 % efficient, and that there is a competitive solute-quenching process. thatfis not independent of A, but that it increases at shorter wavelengths. These results have been recently confirmed by comparisons of the excitation spectra of p-terphenyl solutions in toluene and cyclohexane. The observations on liquid solutions by Brown et aZ.2 (fig. 1-3) show a similar effect at low c, and to a lesser extent at higher c, although these authors conclude that " energy transfer is essen- tially independent of exciting wavelength ".The increase inf has been attributed 2 to increased transfer from the second excited singlet state. This might be due to excitation, through a singlet-triplet transition, of higher triplet states. An alter- native possibility, that cannot be fully eliminated by the experiments to date, is that observed variation offwith X is not a real effect, but an apparent effect associ- ated with the variation of the excitation depth of the solution with A. Dr. F. H. Brown, Dr. M. Furst and Prof. H. Kallmann (New York University) (communicated) : The question of whether some of our results can be accounted for by inhomogeneous solute distribution in plastic samples was raised. This does not appear to be the fact in most cases. An inhomogeneous solute distribu- tion would manifest itself as an unusually large value of Q (energy transfer para- meter) since energy would not be transferred to the solute in regions of low solute concentration 3 and would not be greatly changed at regions of high concentra- tions, It was found, however, that the values of Q in polystyrene are in general similar to those found in liquid solutions, particularly in the presence of naphthalene as intermediate " solvent ".4 Since it does not seem reasonable to assume that energy transfer from naphthalene to solute is better in polystyrene than in liquid solution and since the naphthalene concentration (0.02 M) is so small that it is not likely to influence the solute distribution, it appears that the solute is homo- geneously distributed in most cases. Unusually large Q values are found for fluoranthene and chrysene in poly- styrene (incomplete energy transfer at relatively high solute concentrations). It seems unlikely, however, that of all solutes tried just these solutes are inhomo- geneously distributed in polystyrene.The fluorescence of these solutes show no anomaly in polystyrene under direct light excitation. We believe that the question of whether the peculiar behaviour of these substances can be explained by inhomo- geneous solute concentration is still open. Their anomalous behaviour cannot, however, be explained by differences in the transfer integral of Forster's theory as proposed by Birks because the same solutes behave normally in liquid solvents even when the transferring molecule is the same (i.e. naphthalene).The similarity of the anomalous curves in poly- styrene with and without naphthalene also excludes this possibility. The transfer integral from naphthalene is certainly quite different from that from polystyrene. With regard to Dr. Birks' remarks on radiative transfer at low concentrations, we wish to point out that there is no difficulty in explaining the beginning of our fluorescence against concentration curves. It was pointed out in an early paper on energy transfer in solutions that at low solute concentrations the solute fluor- escence occurs via absorption of solvent radiation by the solute.5 It is only at higher concentrations that the non-radiative transfer takes over. There is no reason to suppose that this is different in a rigid medium.That energy transfer predominantly takes place via a non-radiative process at higher concentrations, in accordance with our early findings, is confirmed by Dr. Birks' own contribution to this meeting. In liquid solutions (toluene + terphenyl) it has been previously reported Brown, Furst and Kallrnann, this Discussion. 2 Birks and Cameron, Proc. Physic. Soc., 1948, 62, 53. 3 Furst and Kallmann, Physic. Rev., 1952, 85, 816. 4 Brown, Furst and Kallmann, J. Chim. physique, 1958, 55, 688. 5 Kallmann and Furst, Physic. Rec., 1951, 81, 853.108 GENERAL DISCUSSION Prof. H. Kallmann (New York University) said: The general shape of the time constant curves of Burton and Dreeskamp as well as those of Knau,l and Swank and Buck 2 can be explained by the theory given by Kallmann and Brucker 3 in which there are essentially two time constants.These constants are rU, the decay time of the solute measured in dilute solutions under light excitation, and T ~ , the decay time of the solvent (which, however, also includes the effect of solute concentration). The equations in that paper are concerned with very short duration of excitation. The calculations have been extended to longer times T of excitation as are applied in Burton’s measurements, and to periodic excitation. For the former, one finds that the rise and decay of a flash contain both time constants, with the shorter time constant that of the less intense component. Since the two time constants occur in the rise and decay curves as the difference of two exponentials, the longer time constant always predominates and the shapes of these curves closely resemble those given by Burton for a terphenyl solution.The shorter time constant shows up only as a bending, as found by Burton. The dependence of the main time constant of terphenyl on the concentration of benzene in cyclohexane as given in Burton’s paper can also be explained according to the theory developed. At zero benzene concentration the measured T is that of terphenyl in cyclohexane since T~ (cyclohexane) is smaller than T ~ . The time constant for energy transfer to terphenyl when benzene is present is the T~ of benzene because that of cyclohexane is very short. In this case, then, in the theory T~ is that of benzene (plus terphenyl) and T~ that of terphenyl in the benzene + cyclohexane combination.The benzene time constant in cyclohexane is larger at small concentrations of benzene than at larger concentrations because of the absence of self-quenching. This, as well as the lower transfer to terphenyl in benzene + cyclohexane com- binations compared to that in benzene alone, leads to a relatively large T ~ , perhaps even longer than ru. This possibility is an explanation of the increase in T found by Burton at small benzene concentration. The curves given by Burton for crystals and plastics seem to be, however, of a different nature, since they present the sum of two exponentials with the longer time constant sometimes that of the less intense component. We believe that this type of curve is not so much a consequence of energy transfer but originates in a quite different process.It has been pointed out in the literature that excita- tion by high-energy particles, especially with a high density of excitation, also results in a longer time constant component. We believe that the crystals and plastic curves presented by Burton are of this type and do not exhibit the effect introduced by energy transfer very clearly since it is probably small. The theory further shows that under periodic excitation even when finite energy transfer is considered a well-defined phase shift will be observed from which T~ and rU can be determined. The results of these calculations are in general agreement with Knau’s measurements. Prof. M. Burton (University of Notre Dame) (communicated): In reply to Prof.Kallmann, from general considerations characteristic of all inter-related first-order decay processes (irrespective of the validity of any theory or postulated detailed mechanism), it is possible to derive a satisfactory equation for intensity of luminescence from an irradiated solution containing a scintillator solute. Using an equation of this type, two decay constants should be calculable from the rise and decay curves-provided the data themselves are perfect. Unfortunately, even our data, which have been determined for a 100-fold variation of intensity, seem inadequate for such determination of two quantities in a single system. Instead, our computation is for the case where one T is dominant. Whether the value determined is for excitation transfer or for scintillator decay is inferred, as 1 Knau, Z.Naturforschung, 1951, lk, 881. 2 Buck and Swank, Argonne National Laboratory, Physics Div., SummaryReport (1 958). 3 Brucker and Kallmann, Physic. Rev., 1957, 108, 1122.GENERAL DISCUSSION 109 in our paper, from other considerations. The bending (cf. region A-B of fig. 1 equivalent to about 10-9 sec) which Prof. Kallmann relates to “ the shorter time constant ” was also so related in our initial (unpublished) considerations. How- ever, the bending must include also the cut-off time of our X-ray tube, also about 10-9 sec. Consequently, the propriety of the assignment which Prof. Kallmann proposes remains to be established. For reasons indicated in the paper, we de- cided against it. The statement that “ at zero benzene concentration the measured T is that of terphenyl in cyclohexane since T~ (cyclohexane) is smaller than T~ ” is not in agree- ment with the totality of facts.Using Prof. Kallmann’s language and our inter- pretation T~ = 2-2 x 10-9 sec and T, is 2.0 x 10-9 sec or less (note the significance of fig. 5). Regarding Prof. Kallmann’s next point, that in mixed cyclohexane and benzene, rs is “ always ” that of benzene, there is an insidious difficulty. To what does rs belong in a solution containing 10-6 vol % of benzene? 10-10 %? A consideration of such extreme cases indicates the snare in the argument. The statement that “ the benzene time constant is larger at small concentration of benzene . . . because of absence of self-quenching ” appears inconsistent with the fact that pure benzene (in which self-quenching should then be extremely high) is an excellent scintillator solvent.Work now in progress in our laboratories sug- gests a somewhat different mechanism. Incidentally, close examination of the succeeding statement by Prof. Kallmann indicates that he has put into words the nature of the curve in fig. 4 but has not provided an explanation. I hope that the beginnings of such an explanation may be presented in a forthcoming paper by Nosworthy, Magee and myself. Regarding the comments on crystals and plastics, there is little chance that the diflerence in behaviour in different cases can be related to density of excitation, which does not vary significantly in these cases. Rather, we are investigating the applicability of a theory of Galanin,l which seems to be more pertinent.Dr. A. Weller (Stuttgart) said: Referring to the last part of his paper, I should like to ask Prof. Terenin how the light-induced proton transfer may be explained with regard to our finding 2 that acridine is about 5 pH-units more basic in the excited state than in the ground state. Prof. A. Terenin (Leningrad) said: The proton photo-transfer studied by us in rigid matrices, as we understand it, does not take place at the excited level of the acridine molecule, but at the ground electronic level. We presume that after the emission act the excess of vibration energy is mainly diverted to the inter- molecular hydrogen bond and is used to overcome the potential energy barrier between the two equilibrium positions of the proton.The change in the fluor- escence spectrum is only a means to observe this transfer which proceeds slowly under illumination by shorter u.-v. light than that exciting the fluorescence, whereas the fluorescence has a lifetime of less than 10-6 sec. Prof. Rufus Lumry (University of Minnesota) said: The comments of several contributors to this Discussion indicate a yearning for the appearance in biological systems of exotic processes depending upon the quantum mechanics of highly ordered systems and the like. While one should not at present dismiss these in any confident manner, it seems desirable not to neglect unusual mechanisms for which we already have evidence and which might be reasonably expected to exist in biological systems. Among these, one of the most intriguing is perhaps the control over protein structure which solvent conditions and substrates may exert. Since in the folding of proteins, and probably in other tightly folded biological polymers, both enthalpy and entropy are lowered, it often occurs that the net free-energy change between conformational isomers of remarkably different descriptions is very small. Interactions with single substrate or inhibitor molecules 1 Galanin, Soviet Physics, JETP, 1955, 1, 317; J. Expt. Theor. Physics, U.S.S.R., 2 Weller, 2. Elektrochem., 1957, 61, 956. 1955, 28, 485.110 GENERAL DISCUSSION throw the equilibrium from one such isomer to another or even favour a nearly total folding or unfolding. A case in point is haemoglobin in which the various additions of oxygen produce marked changes in viscosity, dielectric constant and dielectric relaxation time. The viscosities (fig. 1) were obtained under conditions such that electroviscous effects were very small. As a result they may be attributed to rather large changes in shape, size, or hydration, of the protein molecule which occur through the simple binding of oxygen molecules. Such changes occur only in salt concentrations of the order of 10-3 M or lower and thus may be & I - 100 - 6 0 3 .d Y !!I -- 6 0 2 0 x - 4c Poz(= Hg) FIG. 1.-Intrinsic viscosity of horse haemoglobin (lSo, 5 X 10-5 M KCI) solutions at various degrees of oxygenation (Lumry and Matsumiya, p. 24C, Abstracts of Papers, 134th Meeting, American Chemical Society, Chicago, Illinois, September, 1958). The dipole moment and dielectric relaxation time of these solutions show patterns very similar to that of the viscosity curve (Takashima and Lumry, J. Arner. Chern. Soc., 1958, 80,4238, 4244). of little physiological significance in the reactions of haemoglobin. Nevertheless they do show that very large changes in protein conformation can be produced by simple causes in reactions of small free-energy change. It seems entirely possible that this type of interaction, which could serve for chemical transduction to mechanical effects as in muscle, to changes in membrane permeability as per- haps occurs in vision and to almost every other type of dynamic physical and chemical involvement of proteins in physiological reactions, may be of some real importance in nature. Since such interaction provides a way to couple the con- formational energy of proteins to their chemical reactions, it may even play a role in enzymic catalysis.

ISSN:0366-9033    

DOI:10.1039/DF9592700094

出版商:RSC    

年代:1959

数据来源: RSC

摘要:

IT. ENERGY MIGRATION IN ORGANIZED BIOLOGICAL SYSTEMS INTRODUCTORY PAPER BY ALBERT SZENT-GYORGYI The Institute for Muscle Research, at the Marine Biological Laboratory, Woods Hole, Mass. If a photon, ejected by the sun, interacts with an electron of a molecule on our globe, then the electron is raised to a higher energy level to drop back, as a rule within 10-8-10-9 sec, to its ground state (fig. la). Life has shoved itself between the two processes, catches the electron in its high-energy state and lets it drop back to the ground level within its machinery, using the energy thus released for its maintenance (semi-circle in fig. lb). In order to do this efficiently, life has to meet the photon with a specially suited substance (mostly chlorophyll) and couple this substance to a system which converts the unstable energy of excitation into a stable potential energy.The electronic system which now holds the energy in this stabilized form is symbolized in fig. 1 by the dot, while the oblong and square above it symbolize the possibility of con- verting the primary product into other forms, more fit for storage. The electronic system, in the form of which we take over the energy from the plants, is called by the chemist “fat” or “ carbohydrate ”. A very sketchy and incomplete scheme of how we release the energy from a foodstuff, like lactic acid, is given in fig. 2. As shown by H. Wieland, our foodstuffs are but “ H-donors ”, and as we know from Warburg, lactic acid donates its H’s to a pyridine nucleotide. The nicotinic acid amide, which accepts 3 - 1L i- - A B FIG.1. the H, needs, formally, one H and one electron for its reduction (fig. 2, inset), which means that it can both accept and give off H’s or electrons, can thus act as a sort of exchange counter at which H’s can be exchanged for electrons and vice versa. By the isotope techniques the single H atoms donated to DPN or TPN can actually be identified, 14 but we cannot identify H’s on the next member of the chain, the flavine (FMN in fig. 2). There is thus every reason to believe that what goes from TPN or DPN, step by step, through the rest of the chain of oxidation, are electrons, travelling, in all probability, one-by-one (Michaelis), till they reach oxygen which, after accepting four electrons, turns into H20, capturing protons from the universal solvent, water.The greatest part of the energy of the foodstuff is released in this cascading of electrons from their excited level to their lowest level jn H20, the process which was symbolized in fig. 1 by the half-circle. The energy, thus released, may be invested in the energy of the phosphate bonds of ATP. 111112 INTRODUCTORY PAPER Two problems present themselves at this point. (i) What is the physical basis of this electron mobility? (ii) By what macromolecular organization do biological systems meet this demand for mobility? Several of our speakers will discuss this second problem. I will limit my remarks to the first. Eighteen years ago I asked 11 whether semiconductivity may not underlie the electronic mobility in living systems.Semiconductivity, as a general foundation, failed because in protein the conduction levels are too high and the energy needed to raise an electron into them is not available. Semiconductivity seems to play a major role only in processes in which this energy is available, a photon being involved in the primary process of excitation, as is the case in photosynthesis and vision. In unfinished experiments with Wm. Arnold, we found very strong photoconductivity in flavine, a substance containing extensive systems of conju- gated double bonds. The semiconductivity 8 FAT 11 - C,# 0 f . 0" of such systems demands further study. N R N+ R +FMN+Cytochr. B----+C-+A---jCytochr.Ox.+OZ 5 FIG. 2. 2O My laboratory also studied the possibility of energy transfer between excited molecules.8 That energy can jump from one excited molecule to another over considerable distances was shown by F.Perrin long ago, and we know of many instances of such energy transfer in biological systems (Arnold and Oppenheimer, Bannister, Duysens, G. Weber, Terenin and Ermolaev, Velick and others). All the same, this way of energy transfer also fails as a more general scheme of bio- logical energy transfer, partly because the energy necessary for excitation is avail- able only in processes in which photons are directly involved, and partly because in this way only energy is transmitted, not electrons. During the past years I have been studying, in association with I. Isenberg gb another possibility, the physical basis of which was given by Mulliken's studies on " charge transfer ".In this process the electron is supposed to go spontan- eously from the highest filled orbital of one molecule to the lowest empty orbital of another. A numeric basis for these donor-acceptor relations was given by B. and A. Pullman who calculated the relative energy levels for a great number of biologically important molecules, as shown for a few of them in fig. 3. A biologist, like myself, may be hesitant to build on the result of such involved quantum- mechanical calculations the reliability of which he is unable to judge. We have found7 a simple experimental method to check this reliability and found it sur- prisingly high. We believe now that charge transfer is a very common occurrence in biological systems, even in the ground state, as illustrated by the transfer between tryptophane and flavine or pteridines,sa DPNH and fla~ine.6~ I expect that this transfer will explain not only many instances of electron transfer in biological systems, but will also explain many hitherto obscure pharmacological activities.* * This charge transfer differs from the charge transfer studied by Mulliken in that we find a whole electron going over from the one molecule to the other while in most cases studied by Mulliken only a relatively small fraction was found to be transferred.A .SZENT-GYORGYI 113 Alkaloids, for instance, can act as donors. Charge transfer may be, in fact, one of the most common and important biological reactions. Let us return now to the dot in my fig. lb. Recent work, emanating chiefly from Arnon’s laboratory, suggests that the primary product of photosynthesis is reduction of TPN, and ATP (formed from ADP and phosphate).Formally, the reduction of the pyridine nucleotides is a charge transfer, and may be related to Mulliken’s charge transfer. However this may be, if TPN is the primary stabilization product which corresponds to the dot in our fig. lb, then we can ENERGY OF MOLECULAR ORBITALS HIGHEST FILLED LOWEST EMPTY IMIDAZOLE RYPTOPHANE ACRIDINLORANGE DINITROPHENOLE DPN FIG. 3. pull fig. 1 and 2 together in fig. 4 which shows that the plant cell has a free choice between storing this energy as chemical energy as fat or carbohydrate, or else using it directly as electronic energy for its maintenance. The plant, thus, can live on light-energy more directly than hitherto believed.Carbohydrate, which stood in the middle of our thinking a decade ago seems to become a side-step only. Since we take over the energy from plants in this form we ourselves are also living on light energy in a more direct way than we thought. The chlorophyll-system, naturally, will have to regain its electron, given up in this process, and it seems likely that it will take it from the general solvent, water, which, at the same time is also the final oxidation product of the electron, and thus closes the circle, as symbolized by the long arrow on bottom of fig. 4. Water, the mater and matrix of life, thus would close the circle. Its molecules114 INTRODUCTORY PAPER have very extraordinary properties. As shown by Bernal and Fowler, they easily lend themselves to the building of ordered structures, and so we may ask how far, in biology, we have to do with random or organized water, and it is not impossible that the latter forms an integral part of the machinery of life, facilitating with its structure, also, the transport of electrons or protons, as indicated in fig.4. In any case, the energy of the outer electrons of water are the biological zero level of energy. We may thus picture the energy cycle of life as a periodic boosting- up of these electrons by photons to a higher level from which they return to their zero level in water through the living machinery they drive. The single steps of this circle are the main topic of our present discussions. FAT CnH 0 It jf bFMN4Cytochr.B-+C--bA-+Cytochr.Ox.--+O2 1 TQRe -2- H2° FIG.4. I would like to finish my remarks by a small contribution to the experimental material of this discussion. The investment of the energy released in the oxidative chain into the so-called high-energy phosphate bonds of ATP is what is called " oxidative phosphorylation ". Our observation relates to the question of how the body uses this energy of ATP. Is ATP used as such, as a molecule, or is its energy released in the form of some sort of electronic excitation? In this latter case, one would expect as a first step an uncoupling of electrons within the ATP molecule, one electron being donated by the adenine to the phosphates. This, in its turn, should give a signal in the electron paramagnetic resonance machine. Prof. H.P. Kallmann was kind enough to put ATP in his machine, for us, and found that ATP gives a most interesting signal, an observation which may open new approaches to the understanding of oxidative phosphorylation and the way in which living Nature uses its energy. 1 Arnold and Oppenheimer, J. Gen. Physiol., 1950, 33, 423. 2 Amon, Whatley and Allen, Science, 1958, 127, 1026. 3 Bannister, Arch. Biochem. Biophys., 1954, 49, 222. 4 Bernal and Fowler, J. Chem. Physics, 1933, 1, 515. 5 Duysens, Transfer of Excitation Energy in Photosynthesis (Diss., Utrecht, 1952). 6a Fujimori, Proc. Nat. Acad. Sci., 1959, 45, 133. 6b Isenberg and Szent-Gyorgyi, Proc. Nat. Acad. Sci., 1958, 44: 857. 7 Isenberg and Szent-Gyorgyi, Proc. Nat. Acad. Sci., 1959,45, 519. 8 Karreman and Steele, Biochim. Biophys. Acta, 1957,25,280. Karreman, Steele and 9 Mulliken, J. Amer. Chem. SOC., 1950, 72, 600; 1952, 74, 811 ; J. Physic. Chem., 10 Pullman, B. and Pullman A., Proc. Nat. Acud. Sci., 1958, 44, 1197. 11 Szent-Gyorgyi, Science, 1941, 93, 609. 12 Terenin and Ermolaev, Trans. FaradQy SOC., 1956, 52, 1042. 13 Velick, J. Biol. Chem., 1958, 233, 1455. 14 Vennesland, Fed. Proc., 1958, 17, 1150. 15 Weber, Biochm. J., 1950, 47, 44; Nature, 1957, 180, 1409. Szent-Gyorgyi, Proc. Nat. Acad. Sci., 1958, 44, 140. 1952, 56, 801.

ISSN:0366-9033    

DOI:10.1039/DF9592700111

出版商:RSC    

年代:1959

数据来源: RSC

摘要:

THE SEMICONDUCTIVITY OF ORGANIC SUBSTANCES PART 3.--HAEMOGLOBIN AND SOME AMINO ACIDS BY M. H. CARDEW AND D. D. ELEY Chemistry Dept., Nottingham University Received 14th January, I959 Conductance data have been established for haemoglobin, globin, ferrihaem and a number of amino acids in the solid dry state in zlacuo. Energy-gaps determined by tern- perature variation of the conductance are, for example : globin 2.97 eV, haemoglobin 2-75 eV, ferrihaem 1-74 eV, glycine 2-92 eV, polyglycine 3-12 eV ; ferrihaem falls into the group of molecules, such as phthalocyanine, where conductance is due to n-electrons in the conjugated carbon double bonds. The other substances have a much lower con- ductance, and it is suggested that they are intrinsic semiconductors, due to electron mobility in the CO .. . HN hydrogen bridge system. The relatively small difference between haemoglobin and globin is attributed to the increased degree of denaturation of the globin specimen, with consequent disordering of the H-bridge system. Thermo- electtic power measurements denotes glycine as n-type, and haemoglobin as p-type, the values being small as expected for intrinsic semiconductors. The haemoglobin result may arise from electron trapping by impurities resulting from the heat treatment, or possibly the haems. Conductance measurements along and across the (ac) plane in a single crystal of glycine support the H-bridge theory. The relation of electron mobility to energy gap is discussed and also the implications of the results for three biochemical problems. The electrical conductivity of proteins in the solid state is a subject with im- plications for biochemistry.1s 2 It has been established that both fibrous 3 and globular4 proteins have a definite conductivity in the dry state.The specific conductance K depends on temperature according to the well-known equation for a semiconductor, (1) and the process is associated with thermal excitation of electrons into a conduc- tivity band3 4 Values of the energy gap AE found in these experiments on dry proteins lay in the range 2-2 to 2-7 eV. These values are close to that calculated 5 for excitation of T electrons in the extended hydrogen-bond system ( G O . . . H-N)n, and are much larger than those found for macrocyclic aromatic compounds.4 The present paper describes conductivity measurements on haemoglobin and related component substances such as haem, globin and various amino acids, carried out under controlled conditions in high vacuum.Haemoglobin has a structural relation to several enzymes, including catalase and cytochrome. A brief mention of three of the present results was made at two scientific meetings.69 7 K = KO exp (- AE/2kT), Q-1 cm-1 EXPERIMENTAL The crystals or particles of substances were all examined between platinum electrodes in vacuo, or in some cases with inert gas present to suppress sublimation. The resistances were all greater than 1010 SZ and therefore outside the range of the a.c. method,s and the d.c. method was used,% 8 in which electrodes of area 2.5 cm2 were lightly compressed to 0-5 kg/cm2 on the specimen. However, most of the measurements were carried out in a new " pressure " cell in which platinum electrodes of area 0.11 cm2 were compressed on the specimen with a spring of Ni-Monk 90 at 80 kg/cm2 (h.p.).Some measurements were also carried out at 10 kgfcm2 in this cell, (1.p.). The pressure cell is shown in fig. la. 115116 SEMICONDUCTIVITY OF ORGANIC SUBSTANCES The central electrode is carried on a silica tube to obviate electrical leakage. All cells were wrapped with an earthed screen of aluminium foil or copper gauze. The method was to carry out a series of measurements as the specimen was taken through a number of cycles of heating and cooling, and the maximum rate of temperature change of the cell was 1 deg./min. The first heating always gave anomalously low values due to sorbed moisture, after which very reproducible straight lines of log R against 1/T were obtained.The slope remained constant but the line itself tended to shift to higher resistance values with repeated cycles of heating and cooling. ( td (a1 FIG. 1.-(a) The pressure cell. The upper platinum electrode is carried on a silica tube attached to a ground silica stopper. The outer vessel is of pyrex tube 0.2 in. thick, the flanges being mechanically clamped together on an intervening rubber washer (clamp not shown). A Pt/Pt-Rh thermocouple is brought into the side of the cell via a B7 cone. (b) The thermoelectric power cell. The main part of the cell is shown above, and below is shown the right-hand end of the cell and the gas entry tube.Some measurements of thermoelectric power were made with the cell in fig. l b . The specimen, in the form of a solid piece was lightly compressed between copper electrodes inside a silica tube. The cell was pumped out and filled with 20mm inert gas to assist thermal contact, and contacts between the electrodes and the tube and walls were made with aluminium foil. Two windings enabled a temperature differential to be maintained between the electrodes, usually 15"C, which was alternately reversed in a series of measure- ments. A third winding enabled the average temperature to be maintained at about 160"C, where specimen resistance values were in the range 1011 to 1012 Q. In measuring the thermoelectric e.m.f. one electrode was earthed and the other connected to the grid of an ETl electrometer valve in a valve voltmeter.Thermocouples measured the tem- perature differential. Heaters were non-inductively wound and run off accumulators, and the silica tube was covered with an earthed screen of aluminium foil under the elec- trode windings. The accuracy of the thermoelectric e.m.f. results was limited by the unusually high resistance values of the specimens examined. PREPARATIONS HAEMOGLOBIN (FERROHAEMOGLOBIN) Red cells were separated from fresh ox blood, washed four times with 0.9 % NaCl solution, followed by dialysis, first against tap water then against distilled water, in a cold room. The stroma were removed by addition of ether followed by centrifugation. The lower layer containing the haemoglobin was sucked off, and ether and oxygen pumped off in a desiccator, a trace of silicone being added to prevent frothing (solution A).Haemo- globin (I) was separated as a brittle gel by evaporation of solution A at 5°C over CaC12 in a desiccator. Many measurements were made on haemoglobin precipitated by addition of methanol at room temperature, referred to as methanol denatured haemo- globin (11). Oxyhaemoglobin crystals were prepared by bubbling oxygen through solution A, then adding 25 % cooled ethyl alcohol at - 10°C and allowing to stand for some hours.9M. H. CARDEW A N D D . D. ELEY 117 These crystals on standing in their mother liquor for several months autoxidized to methae- moglobin (ferrihaemoglobin), which could be separated and dried between filter papers so long as the temperature did not rise above 0°C.GLOBIN.-Denatured haemoglobin was extracted with methanol for 24 h in a Soxhlet extractor, which dissolves the haems, leaving behind the globin. The haem was recovered from the methanol solution as haematin (ferrihaem). POLYGLYCINE, D.P. 150-200, was supplied by Dr. C . H. Bamford in the form of a powder. THE AMINO ACIDS examined were all commercial preparations, purified by recrystalliza- tion from distilled water or water + methanol mixtures. RESULTS THE ENERGY GAP IN HAEMOGLOBIN AND GLOBIN A series of runs were carried out on methanol denatured haemoglobin and on globin and haem separated from haemoglobin : some of the results are shown in fig. 2, 3,4, where the parallel lines correspond to different cells and various thicknesses of specimen in the same cell.The results are summarized in table 1. 1 0 3 i ~ FIG. 2.-Conductivity results for haemoglobin. A run on native haemoglobin in the form of a piece of dry gel gave he = 2.8 eV. The energy gap is not affected by a seven-fold change in the voltage applied across the specimen. The effect of raising the applied pressure from 0 to 80 kg cm* is possibly TABLE ENERGY GAPS AT 400°K pressure voltage A€, eV A log R* globin haemoglobin cell kglcmz V normal 0.5 16.5 3.02 2-74 1.0 pressure I0 16-5 3.18 2.94 1-4 16.5 2.82 2.66 0.8 80 120 2-87 2-58 1.1 av. 2.97 2.75 1.1 - - 2.82 Y 9 10 120 7 7 80 9 9 - - - * The difference in the logarithm of the resistance values of globin and haemoglobin at 400°K.118 SEMICONDUCTIVITY OF ORGANIC SUBSTANCES to lower A€ by 0-2 eV, but this was not definitely established and in any case is much smaller than for phthalocyanine and isodibenzanthrone crystals.4 It has been considered best to average the A€ values for the purpose of table 2.There is a ten-fold increased specific resistance of globin over haemoglobin. 14.0 Qa 13.0 e B 0 0 4 12.0 I I I I 0 0 2-3 2.4 2.5 1 0 3 1 ~ FIG. 3.-Conductivity results for haem. 2-3 2.4 2.5 2.6 2.7 1031~ FIG. 4-Conductivity results for globin. THE SPECIFIC RESISTANCE AND THE ENERGY GAP Specific resistance values for the powders were calculated by the equation p = RAV/I, 51 cm, where R is the measured resistance of a powder specimen area A , thickness I (usually about 0.15cm) and volume fraction V. For powders in the normal cell we measured V as 0.39, and for the pressure cell we assumed close-packed spheres with V = 0.74.M .H . CARDEW AND D . D . ELEY 119 Methanol denatured haemoglobin at 400°K gave p = 5.3 x 1012 52 cm and 5-8 x 1012 52 cm in these two cells, while native haemoglobin in the form of a flat piece of dry gel aluminized on two sides gave 2.7 x 1012 .Q cm. Globin at 400°K in the pressure cell gave 5.3 x 1013 SZ cm and a single piece in the normal cell gave 3.8 x 1013 52 cm. The three haemoglobin values and the two globin values are averaged in table 2. substance 1. haemoglobin 2. globin 3. haem 4. a-alanine 5. /3-alanine 6. glycine * I ac 7. glycine 11 ac 8. glycine 9. DKP 9 , 9 , 9 9 10. oxamide 11. tyrosine 12. polyglycine * 13. glycine C.C. TABLE 2.-cONDUCTNITY DATA TOK P, Q cm 400 4-6 x 1012 400 1.3 x 1012 400 4 3 x 1013 > 405 5.3 x 1014 < 405 5.3 x 1014 > 400 6.4 x 1013 < 400 6.4 x 1013 > 400 1-8 x 1013 < 400 1.8 x 1013 400 5.3 x 1012 400 1.7 x 1012 > 430 1.3 x 1016 (extrap.) (extrap.) 430 2.7 x 1015 430 1.1 x 1015 400 2.0 x 1013 400 1015 AE eV 2.75 2.97 1 *74 3-31 2-16 4-07 3.12 2.67 2.82 1-99 2.92 2-19 1-87 (2.2) 3.12 - log q(R-1 cm-1) 4.6 5.0 6.1 2-8 12-9 5.1 2.3 3.7 2.5 6.1 5.6 2.8 4.3 2.8 6.3 * All p values are at 400"K, being extrapolated where necessary, e.g.for 9, 10, 11. A€ and KO refer to 400°K or to the temperature range in the first column. An asterisk denotes use of a single crystal or piece of the substance. DKP, diketopiperazine; glycine C.C., glycine copper chelate. The only crystalline protein examined was methaemoglobin and several runs at 10 and 80 kg/cm2 in which the protein was kept below 110°C gave Ae = 2.5 eV.Over a higher temperature range, 400-450°K, A€ = 3-1 eV and log KO = 4.8. It is possible that the former values may indicate a somewhat lower value for the native haemoglobin molecule, and after heating to 110°C the protein still showed some solubility. However, a more recent measurement by D. Spivey, also below 110°C gave AE = 2.66 eV and log KO = 4.34 for a powdered dry gel of native ferrohaemoglobin at 70 kg/cm2. Further work is in progress on this problem. EFFECTS OF IMPURITIES For the proteins the data referred to specimens made salt-free by dialysis. Addition of about 1 % NaCl to haemoglobin led to a characteristically different behaviour. The log R against 1/T curves differed markedly for heating and cooling and their slope gave Ac < 2.75 eV.Dry air had no measurable effect on the conductivity of a piece of dry gel of native haemoglobin. The dry proteins showed no measurable decomposition on heating in vacuo up to lWC, although they became insoluble in water and therefore denatured to some extent. Haemoglobin showed a measurable gas evolution, starting at 125°C. For a methaemoglobin sample log (dpldt) against T-1 obeyed the Arrhenius law with 2E = 3.0 eV, which is close enough to the energy gap of 3.1 eV to suggest de- composition is initiated by an electron (or hole) reacting with a particular side chain. Temperature-cycling between 25°C and 125°C had no noticeable effect on Ae but the resistance at a given temperature showed a small continuous increase with time.At 180°C this increase in resistance of haemoglobin with time was easily followed. At 200°C the protein became plastic, but there was no discontinuity in behaviour, except in the pressure cell, where the compression caused a decrease in specimen thickness which showed up as a sudden decrease of resistance.1 20 SEMICONDUCTIVITY OF ORGANIC SUBSTANCES THE AMINO ACIDS Glycine was examined as a single crystal between platinum electrodes and measure- ments were made parallel and perpendicular to the ac plane which contains the zwitterions hydrogen-bonded in two-dimensional sheets. Polyglycine powder was melted to form a single piece of film. All the other substances were examined at 80 kg/cm2 in the pressure cell.Where necessary the vacuum was replaced by 10 mm nitrogen to reduce sublimation. Glycine and a-alanine each gave two straight lines for log R against 1/T meeting in a discontinuity around 400°K. The glycine results are shown in fig. 5, and it may be assumed that an impurity mechanism operates below the discontinuity and an intrinsic 1 0 3 / ~ ---- , -O-O-, -O--O--: across H-bonded layers. FIG. 5.-Conductivity of a glycine single crystal. - x x x -: along H-bonded layers. mechanism above it. Polycrystalline glycine also showed this discontinuity but at 370°K so the value of A€ quoted is for the intrinsic range. Crystals of diketopiperazine, oxamide and tyrosine were so little conducting that measurements were not made below 430°K.The log R against 1/T results did not lie on a straight line for tyrosine, so the A€ value quoted is very rough. Although glycine copper chelate showed a little conductance at high temperatures it was always subsequently found to have decomposed to some extent. THERMOELECTRIC POWER These measurements were difficult to carry out. A methanol denatured specimen of haemoglobin was dried out to give a solid gel, and a specimen of this, 0-7 mm thick, was aluminized on each side. Each value was the mean of 5 or 6 readings in which the thermal gradient was reversed. One specimen gave a p-type thermoelectric power of 48 3 pV/deg. (162°C) and 500 pV/deg. (158°C). The specimen was reversed and then gave 610 pV/deg. (165°C). The resistance of this specimen was about 1011 In and it showed an asymmetry potential of 200 mV, unaffected by temperature or temperature gradient, which reversed as the specimen was reversed.For glycine measurements were made on a 1.0 mm thick wafer split from a single crystal along the uc plane. Glycine gave an n-type thermoelectric power, - 360 pV/deg. (153°C) and - 730 pV/deg. (166°C). The effects of temperature are probably not significant. With haemoglobin the temperature used of 160°C must certainly have resulted in a certain amount of decomposition. The observed p-type character may well have resulted from trapping of electrons by the decomposition products and until we have made further studies at lower temperatures, we shall take the glycine results as characteristic for hydrogen-bonded systems.M.H . CARDEW A N D D . D . ELEY 121 DISCUSSION PHYSICAL ASPECTS 1NTERGRANULAR RESISTANCE While the high frequency a.c. method is to be preferred for conductivity measurements, the high resistance of dry proteins and amino acids compels the use of the d.c. method. Despite this, the resistance of the intergranular boundaries should be negligible by comparison, especially when the specimen is under com- pression. Where checks were made between compressed powder and a solid piece of dry gel, or single crystal, specific resistance values agreed to a factor of 2 and energy-gaps to about 0.2 eV. IONIC AND ELECTRONIC CONDUCTIVITY On application of the potential the current through the substances examined in this paper reaches a steady value in a short time, about 1 min, and this supports the earlier view 3 . 4 that the observed conductance is a true electronic semi- conductivity.Resistance values and energy-gaps were very similar for different haemoglobin preparations (a result further confirmed by D. Spivey in this labor- atory). The addition of 1 % NaCl which might be expected to give an ionic conductance, gave rise to characteristically different log R against 1/T curves for haemoglobin. The effect of decomposition of haemoglobin at 180°C was to give a lower conductivity, whereas a higher conductance would be expected if due to ionic impurities, especially when the specimen became plastic at 200°C. Instead the introduction of decomposition products in lowering the conductance may plausibly be explained in terms of the production of trapping sites for current carriers, probably electrons.Proton conductivity has been postulated for poly- amides with NH . . . 0 hydrogen bonds (A. = 2.58 eV) 11 and for certain OH . . . 0 hydrogen bonded carboxylic acid (A, = 0-5 to 6 ev) 12 and it is interesting that both these systems have specific conductances and KO values 103-105 times those found here. Polyamides will be the subject of a further paper from this laboratory.13 It seems probable that adsorbed water will induce a proton conductivity in solid proteins. We have recently completed a study of the adsorption of water on haemoglobin,l4 and the conductivity of this system is being examined. HYDROGEN BONDS AND CONDUCTANCE Evans and Gergely 15 have calculated that repeat units of the kind (NH .. . 0 = C) will lead to three narrow energy bands, width about 0.2eV, the lower two filled with electrons and the upper one empty. A model in which trigonal sp2 bonds are assumed for the N atoms, with an N-0 distance of 2.65A, leads to an energy gap of 3.05 eV between the top of the filled and the bottom of the unfilled bands. Such hydrogen-bonded repeat units exist between parallel poly- peptide chains in the /3-protein configuration found in stretched fibres, and along the spiral of a single polypeptide chain in the a-configuration, in globular proteins,l6 and also in the crystals of those amino acids which have been examined. The energy bands arise from the collective nature of the pz or n electrons on the N, 0 and C atoms, and it is interesting that the observed energy gaps for both globin and polyglycine are close to the calculated value, although the N-0 distance in most cases is probably 16 about 2.72& somewhat larger than that used in the calculation.Similar values hold for glycine in the intrinsic range (400"K), both in the polycrystalline form and as single crystals. The closely similar behaviour of glycine and globin rule out a mechanism based upon mesomeric changes in the polypeptide backbone as postulated by Denbigh.17 The results on glycine single crystals appear to support the view that there is a slightly higher conductance and lower energy gap parallel to the plane of the H-bonds. Glycine crystals122 SEMICONDUCTIVITY OF ORGANIC SUBSTANCES are monoclinic and consist of continuous sheets of planar zwitterions, each NH3f forming one bond of 2.76 8, and one of 2.88 8, to neighbouring 0- ions.18 The energy gap for polycrystalline glycine is intermediate between the " parallel " and " perpendicular " single crystal values in agreement with expectation.Diketo- piperazine 19 and oxamide 20 are hydrogen-bonded in chains, in a similar fashion to protein structures. DL-Alanine has a three-dimensional hydrogen-bonded structure.21 All give conductance values less than for globin, although the energy gaps vary in no regular fashion. /3-Alanine shows a conductance similar to haemoglobin although its energy gap is very high. The extra CH2 inserted in the chain of NH . . . O=C bonds has not exerted any marked " insulating effect ". The tyrosine result indicates that a phenyl group does not introduce any extra conductivity in the glycine system, and provides evidence that the side-chain of amino acids are not responsible for the semi-conductivity.The similar be- haviour of polyglycine and globin also support this view. Confirmation of the role of (NH . . . O=C) bonds comes from the lack of conductivity in glycine copper chelate, where the normal arrangement of hydrogen bonds must be changed from glycine itself. A value of about 3.0 eV may be fairly commonly found for proteins. Recent work by D. Spivey in this laboratory has given a value of 3.0 f 0.13 eV for pig insulin, and further proteins are under examination. The thermoelectric power results, although rough, support the view that glycine crystals are intrinsic semiconductors, in which the electrons show a larger mobility than the positive holes, which is normal behaviour.If glycine were an n-type impurity semiconductor we should expect a thermoelectric power of order - AQeT, seven times that observed. The ratio of electron to hole mobility is calculated as b = 1.33 from the following equation 22 Unfortunately we have not yet been able to measure the thermoelectricity power of globin. THE PROSTHETIC GROUP Crystalline ferrihaem shows an energy gap, 1-74 eV which is very close to that found for metal-free phthalocyanine 8 of about 1.5 eV, and its behaviour puts it in the same class of intrinsic n-electron semiconductors. Our view here is that AE measures the energy required to promote an electron from the highest filled to the lowest unfilled (possibly triplet) molecular orbital, from which it may tunnel from molecule to molecule throughout the crystal.The relatively high conduc- tivity of the porphyrin molecules are lost on combination with globin. The ob- served energy gap for haemoglobin is so much greater than 1.74 eV that electron tunnelling between haems is very unlikely, and this view is supported by estimates of the spacing between the centres of the haem groups. If the haems are uniformly distributed through the solid, as possibly in highly denatured haemoglobin, their spacing will be 27A. The orientation of the haems in natural haemoglobin has recently been decided by electron-resonance work.23 They are known to lie in two groups of 2, separated by 64" and facing the haems in neighbouring molecules. Assuming the haems are on the surface of the molecule, a maximum haem separa- tion of 248, may be estimated.These two spacings are much larger than the haem separation in ferrihaem crystals, and make a direct electron tunnelling between haems rather unlikely. The conductivity of haemoglobin is ten times that of globin at 400°K and the energy is 0.23 eV lower, and this difference has been definitely established by com- parative measurements under various conditions. The p-type behaviour of haemo- globin may indicate either (i) that haemoglobin is an intrinsic semiconductor withM . H . CARDEW AND D . D . ELEY 123 holes more mobile than electrons (b = 0.84 using the above equation), or (ii) that it is an impurity semiconductor with holes as majority carriers. Considering first (ii), it has not proved possible to construct a consistent scheme in which the haems function as impurity centres, accepting electrons from the conduction band of the protein.The thermoelectric measurements of necessity were carried out at 16OCC, and it seems possible that thermal decomposition products were acting as electron traps and reducing electron mobility in this way, accounting for the p-type behaviour. Preference is therefore given to theory (i) that haemoglobin is an intrinsic semiconductor due to rr-electron excitation in the C=O . . . HN bridge system. The process of extracting methanol-denatured haemoglobin is bound to result in a further disordering of the polypeptide chains and hydrogen bridges in the resultant globin.So it is likely that the decreased conductivity is associated with a disordering of the hydrogen-bridges in the globin. It is possible that the haems themselves act as electron-traps in haemoglobin thereby helping to make it p-type. Such a role for the haems is supported by the fact that single crystals of the chemically similar phthalocyanine show p-type be- haviour .24 For an intrinsic semiconductor the number of charge carriers per cm3 is given by The factor 2 in brackets is to be included for globin and haemoglobin since in each case the highest filled molecular orbital may be supposed to hold 2 paired electrons. It is necessary to assume the effective mass of the average charge carrier YIP is that for the free electron. Given n, and e the electronic charge, the value of p, the average mobility for holes and electrons is obtained from the observed conductance K, with results below, K = nep.Globin, 400"K, n = 21 per cm3, ,u = 7 x 104 cm2 per volt sec. Haemoglobin, 400"K, n = 508, p = 2.3 x 104 cml per volt sec. These p values agree with the proposal that the haems in haemoglobin act as electron traps, and thereby lower the average mobility. The above calculation may8 be put into the form KO = (2)4p at 293"K, or using different assumptions Many, Harnik and Gerlich 25 give the equation KO = (2)16p. The above two values of p are very high and are only equalled by a few inorganic semiconductors such as TnSb. On the other hand it was pointed out that certain velectron aromatic compounds had very low KO values, and therefore very low p values.8 Many, Harnik and Gerlich 25 reviewing the published data for aromatic compounds show that KO, and p cal- culated from it as above, increases parallel to the energy gap A€.Fig. 6 shows that the present results demonstrate a rough linear relationship between log KO, and therefore log p and A,. The numbers on the points are those given in table 2, and two points for insulin are included. The full line through these points is given by loglo KO (SZ-1 cm-1) = 8 A, (eV) - 18.9 and from this the equation for loglop is easily calculated. The horizontal band contains most of the values for elemental inorganic semiconductors, and it may be seen that the present results fall above and below these values. It is of interest to compare these results with the data for aromatic rr-electron semiconductors, and this has been done for all established values, with the follow- ing results.Points are omitted to avoid confusion in the diagram. The best values for more highly conducting aromatic substances are probably given by the a.c. high-frequency method, so far as polycrystalline specimens are concerned. The dotted line is drawn through points for diphenyl picrylhydrazyl,124 SEMICONDUCTIVITY OF ORGANIC SUBSTANCES isodibenzanthrone, and metal-free phthalocyanine obtained by this method. This line probably represents the upper limit for loglo KO for a given A€, and its equation is loglo KO (52-1 cm-1) = 6 . A€ (ev) - 8, It would be expected that d.c. single-crystal data would lie near this line, and this is so for phthalocyanine single crystals.24 Most of the other d.c.data on organic substances, such as the polycrystalline studies of Inokuchi 26 and the single-crystal studies of Northrop and Simpson 27 and Pick 28.29 lie scattered in between the full and dotted lines. This includes the present value for ferrihaem. It seems likely that these data are all subject to Ar, eV FIG. 6.-The observed relation between log KO and A€ for proteins and amino acids numbered as in table 2. No. 13 and 14 refer to pig insulin, measured by D. Spivey. The dotted line is drawn through previous data of Dr. G. D. Parfitt (points not plotted) obtained by the high frequency a.c. method on isodibenzanthrone, metal-free phthalo- cyanine and diphenyl picryl hydrazyl. - - - -, rr-molecules a varying extent to the effect of intergranular resistances, in lowering logloKO or raising A€.An exception to this is the recent d.c. data on phthalocyanine powders which lie somewhat above the dotted line.30 As remarked earlier, Eley and Parfitt associated AE for an aromatic molecule with excitation of an electron from the highest filled to the lowest unfilled molec- ular orbital, from which it may tunnel, under the applied voltage gradient, to the next molecule, the tunnelling process determining the mobility p and therefore KO. Many, Harnik and Gerlich 25 also adopted this view, pointing out the implication that the excited electron in a compound with high AE will tunnel more easily andM. H . CARDEW AND D . D. ELEY 125 will therefore have a higher p and KO.This view may now be applied to the velectrons in the C=O . . . H-N system, where the barrier will lie between the 0 and H atoms and will have a different character to that between two aromatic molecules. Therefore it may be understood that the two loglo~o against AE lines have a different slope. It is hoped to develop this model on more quanti- tative lines. Actual calculations of mobility p depend on values of n, which in turn depends on assuming m* is the free-electron mass. In proteins, where the energy bands are narrow, m* may be more than the free-electron mass. BIOCHEMICAL ASPECTS There is a very small but quite definite semiconductivity associated with pro- teins and amino acids in the dry state. Thus the extrapolated resistivity of the methanol denatured proteins are globin 1019, haemoglobin 4 x 1017 Q cm at room temperature, and their respective energy gaps 2.97 and 2-75 eV.The increase in resistivity and energy gap from haemoglobin to globin may be associated with an increased degree of denaturation and corresponding disorientation of hydrogen bonds. On this view we might expect native haemoglobin to have a lower energy gap than 2.75 eV, and values of 2.66 eV for haemoglobin and 2-5 eV for methaemo- globin have been recorded in runs up to 110°C only. To establish the reality of such differences will require further comparative measurements, but in any case the effects are small. Dry insulin behaves similarly to haemoglobin and globin, and its seems reasonable to base some general con- siderations on the haemoglobin data.In nature a protein in a lipoidal environment might approach the anhydrous condition, but probably never reach it. The observed semiconductance is relevant to three problems in biological systems. (i) It has been conjectured that protein systems may conduct electrons between cytochrome systems in the cell.2 As an example, the respiration rate of a sea urchin egg of 3.5 x lO--Cc.mm. s.t.p. of oxygen per hour corresponds to an electron current of 16.8 x 10-11 A, which, taking a potential gradient of 1 V as typical of redox systems, could be carried by a fibre of resistance 0-6 x lOlOQ. However, a model fibre of haemoglobin molecules 1 p long by 50A thick would have a resistance of 2 x 1026 Q, which is much too high to carry the current.In any case, the apparent activation energy would be determined as half the energy gap, i.e. 32 kcal compared with the usually observed values for respiration of about 11 kcal/mole. Electrons liberated with this excitation energy might enter the conduction band, of course, when their high mobility would imply a mean free path of 4p, adequate to traverse a mitochondrion, for example. This does not seem a likely mechanism, however, in thermal reactions. (ii) There seems a definite possibility for photoexcitation of electrons into the conduction band by any quantum greater than about 3 eV. Such a quantum if absorbed would give rise to an electron and a positive hole, which migrating together would constitute an exciton capable of transferring energy and causing chemical reaction in distant parts of the molecule or system of protein molecules.Thus wavelengths in the range 1960-3660 A are classically cited 1.31 as equally effective in the inactivation of urease.32 Since 3660 8, corresponds to a quantum of energy 3.39 eV, if the enzyme urease behaves like the proteins here discussed, this energy should be more than adequate to create an exciton. Similarly, light of 28008, absorbed by tryptophane and tyrosine residues in the globin moiety, can cause dissociation of carbon monoxide from the iron atom in the haem part of carbon monoxyhaemoglobin.33 Since this quantum is 4.43 eV it is more than adequate for exciton formation. Where the electron and hole migrate in opposite directions under an applied or " built-in " electric field we have effectively the generation of reducing and oxidizing power at the two ends of the hydrogen-bridge system.Thus any chemical126 SEMICONDUCTIVITY OF ORGANIC SUBSTANCES change removing an H atom from an NH group is an oxidation and equivalent to removal of an electron from the valence band of our model. The positive hole left in the valence band may then migrate to the terminal 0 atom of the system where it will act as oxidizing agent (electron acceptor). This may be expressed in terms of a keto-end change as was done by Geissman34 and Schmitt35 (cf. a review, ref. (36)). I I I I I I J . I I I H.+ *N-C=O . . . H-N-C-0 . . . H-N-C-0 N=C-O-H . . . N=C-0-H . . . N=C-Ote. (iii) It is a matter of interest to enquire how far a conjugated substrate or coenzyme can extend its resonance system by combination with a specific protein.Thus dehydrogenation has been supposed to involve as rate-determining step formation of a semiquinone radical, and the function of a specific protein may be the resonance stabilization of this radical.37~ 38 (Recently there has been found electron-resonance absorption evidence for the stabilization of semiquinone by inorganic surfaces 39.) The effect of pH on the dissociation curve of oxy-haemo- globin involves transmission of mesomeric effects over only two or three bonds of the imidazole group to the Fe atomPo but the interaction effects between the four haems in a molecule 41 must involve transmission through many amino-acid residues. Spectroscopic evidence on the other hand suggests that mesomeric inter- actions are short range in proteins.As a standard for comparison, the addition of one (CH=CH) unit to a carotenoid structure shifts 42 its absorption maximum roughly 300 ?L towards the red 41 and for polymethene dyes 43 the shift is 1100 A. In haemoglobin the shift must be negligible, since in haem the broad band stretches from 540-580 mp, compared with the absorption maximum 44 in haemoglobin of 559 mp. When alloxazine is bound to its specific protein its absorption maximum shifts only 200 A to the red 45 and a similar shift occurs for chlorophyll as when bound in chloroplast.46 Thus the effect of a specific protein in extending the resonance system corresponds at most to one carbon-carbon double bond. Our conductivity results lead us to state earlier that " mesomeric paths in proteins are probably limited to the immediate neighbourhood of the active site and in this respect proteins probably differ from semiconducting oxide catalysts ".7 If the haemin system had been extended throughout globin we should have expected haemoglobin to have an energy gap of 1.74 instead of 2-75 eV.In fact, in the model advanced earlier in this paper, effectively the haem molecules form con- ducting centres embedded in the relatively insulating globin matrix. Hence the quoted conclusion, which agrees with the spectroscopic results. The basic reason for the energy gap of 2.7 eV in proteins is of course the relatively small N . . . 0 overlap integral of - 0.20eV as assumed by Evans and Gergely.5 We should not expect a strong n-interaction between this system and one involving con- jugated carbon atoms with the much higher overlap of - 0.87 eV.The con- ditions for interaction of two n-electron systems have been discussed by Huang and Wylie.47 There remains the necessity for explaining protein-prosthetic group inter- actions such as those giving rise to the chemical differences between haemoglobin, catalase, peroxidase and cytochrome-c.48 Here haem is linked to four different proteins which must exert specific effects on the central iron atom. Further semi-conductivity work is desirable on these systems, but on the present view the chemical differences are to be explained in terms of short distance mesomeric interactions, such as already postulated to explain the pH effect in haemoglobin 40 and possibly also by steric effects of the protein on the central Fe atom.It is known that haemoglobin on drying at 25°C in vacuu gives anhydrohaemo- globin, which is still soluble in water.49 It is known that water is necessary forM. H. CARDEW AND D. D. ELEY 127 the dissociation of oxyhaemoglobin, and it may be possible therefore that oxygen will not react with dry haemoglobin, so the absence of an observed conductivity change is not decisive. Furthermore, we have not established any definite effect of the oxidation state of the metal atom on the semiconductance. It is known for phthalocyanines that the presence of various metals has only a secondary effect on its semicond~ctivity.24~ 30 If the effect of haem itself is so small on the conductivity of globin, even smaller effects would be expected for changes occurring at the Fe atom.Barron 50 found that alkaline denaturation shifts the redox potential of haemoglobin by 0-07V. If this effect were due to a disordering of the H-bridge system effecting the ionization potential of the central Fe atom, then some effects of the central atom should be detectable on the semiconductivity. The result, however, may arise simply from an N atom displacing H20 from the Fe atom in the denaturation process. It is possible that water adsorbed on protein molecules may give rise to new electronic effects at least in the surface of proteins. Baxter 3 postulated adsorbed water acted as impurity donor centres in wool fibres. This matter is being studied for haemoglobin in our laboratory.The observed time effects should serve to distinguish electron conductivity, from proton mechanisms of the Grotthus-type, postulated by WirtqS1 which may be more prominent than for dry proteins. We are also studying the photoconductivity behaviour of proteins. Rittenberg and Krasna 52 have referred to the possible significance of a triplet state in proteins for binding substrate for enzymes. Eley 6 has suggested that the semiconducting state may be the excited state concerned. Anslow and Nassar 53 have assigned an absorption maximum of 2800A to peptide bonds in proteins, corresponding to an energy of 4.4 eV which is much higher than the semiconductivity energy gap. This matter may possibly be resolved by the photoconductivity work, which is being undertaken in this laboratory.The authors gratefully acknowledge a grant from the D.S.I.R. (Food In- vestigation) to M. H. C. which made this work possible. 1 Moglich and Schon, Naturwiss., 1938, 26, 693. 2 Szent-Gyorgyi, A., Nature, 1941, 148, 157 ; Bioenergetics (Academic Press, New 3 Baxter, Trans. Faraday SOC., 1943, 34, 2071. 4 Eley, Parfitt, Perry and Taysum, Trans. Faraday SOC., 1953, 49, 79. 5 Evans and Gergely, Biochim. Biophys. Acta, 1949, 3, 188. 6 Eley, Farnday SOC. Discussions, 1955, 20, 273, 282. 7 Eley, Advances in Catalysis, 1957, 9, 372. 8 Eley and Parfitt, Trans. Faraday Soc., 1955,51, 1529. 9 Keilin and Hartree, Proc. Roy. Soc. By 1935, 117, 1. 10 Eriksen, Nature, 1954, 173, 727. 11 Baker and Yager, J. Amer. Chem. Soc., 1942, 64, 2164.12 Pollock and Ubbelohde, Trans. Faraday Soc., 1956, 52, 11 12. 13 D. Spivey, to be published. 14 Cardew and Eley, Dehydration Conference (S.C.I.), to be published in J . SOC. Chem. 15 Evans and Gergely, Biochim. Biophys. Acta, 1949, 3, 188. 16 Pauling, Corey and Branson, Proc. Nat. Acad. Soc., 1951, 37, 205,235. 17 Denbigh, Nature, 1943, 154, 642. 18 Albrecht and Corey, J . Amer. Chem. SOC., 1939, 61, 1087. 19 Corey, J. Amer. Chem. Soc., 1938, 60, 1598. 20 Ayerst and Duke, Acta Cryst., 1954, 7, 588. 21 Levy and Corey, J. Amer. Chem. SOC., 1941, 63, 2095. 22 Johnson and Lark-Horovitz, Physic. Rev., 1953, 92, 226. 23 Bennett, Gibson and Ingram, Proc. Roy. SOC. A , 1957, 240, 67. 24 Fielding and Guttmann, J. Chem. Physics, 1957, 26,411. 25 Many, Harnik and Gerlich, J. Chem. Physics, 1955, 23, 1733. York, 1957). Ind.128 SEMICONDUCTIVITY OF ORGANIC SUBSTANCES 26 Inokuchi, Bull. Chem. Soc. Japan, 1952, 24, 222 ; 25, 28. 27 Northrop and Simpson, Proc. Roy. Soc. A, 1956,234, 122. 28 Mette and Pick, Z. Physik, 1953, 134, 566. 29 Pick and Wissmann, Z. Physik, 1954, 138,436. 30 Felmayer and Wolf, J. Electrochem. Soc., 1958, 105, 141. 31 Jordan, Naturwiss., 1938, 26, 693. 32 Kubowitz and Haas, Biochem. Z., 1933,257, 337. 33 Bucher and Kaspers, Biochim. Biophys. Acta, 1947, 1, 21. 34 Geissman, Quart. Rev. Biol., 1949, 24, 309. 35 Schmitt, Z. Naturforsch., 1947, 26, 98. 36 Bucher, Adv. Enzymology, 1953, 14, 1 (Interscience, N.Y.). 37 Michaelis, Ann. N. Y. Acad. Sci., 1940, 40, 39 ; Amer. Scientist, 1946, 34, 73. 38 Kalkar, Chem. Rev., 1941, 28, 72. 39 Bijl, Kainer and Rose-Innes, Nature, 1954, 174, 830. 40 Coryell and Pauling, J. Biol. Chem., 1940, 132, 769. 41 Pauling and Coryell, Proc. Nut. Acad. Sci., 1936, 22, 159, 210. 42 Bayliss, J. Chem. Physics, 1948, 16, 287. 43 Sklar, J. Chem. Physics., 1942, 10, 521. 44 Lemberg and Legge, Haematin Compounds and Bile Pigments (Interscience, N.Y., 1949). 45 Haas, Biochem. Z., 1937,290,291. 46 Rabinowitch, Photosynthesis (Interscience, N.Y., 1945), vol. 1, p. 383. 47 Huang and Wylie, Faraday Soc. Discussions, 1950, 8, 18. 48 cf. Theorell, Adv. Enzymology, 1947, 7, 265 (Interscience, N.Y.). 49 Haurowitz, J. Biol. Chem., 1951, 193, 443. 50Barron, J. Biol. Chem., 1937, 121, 285. 51 Wirtz, 2. Naturforschung, 1948, 36, 131. 52 Rittenberg and Krasna, Faraday SOC. Discussions, 1955, 20, ! 85. 53 Anslow and Nassar, Opt. Soc. Amer. J., 1941, 31, 114.

ISSN:0366-9033    

DOI:10.1039/DF9592700115

出版商:RSC    

年代:1959

数据来源: RSC

摘要:

CHARGE TRANSFER PROCESSES IN BIOLOGICAL SYSTEMS BY R. MASON Dept. of Chemistry, University College, London Received 20th January, 1959 The biological effects of radiation are discussed in relation to long-lived excited states, involving v electron rearrangements, in proteins. The increase in radiosensitivity which oxygen and nitric oxide confer on a number of biological systems is attributed to their perturbation of singlet-triplet transitions while the radiobiological effects of the inert and a number of other gases may derive from their collisional deactivation of the triplet states. The specificity of action of aromatic hydrocarbons is found to be paralleled by the probability of a charge-transfer complex representing a transition state in the reaction of these molecules with proteins ; nucleic acid derangements, allowing the transmission of modified " information ", may follow the absorption of excitons produced through electronic degradation processes.The semiconductor description of a protein 192 depends essentially on the assumptions that intramolecular hydrogen bonding allows an interaction between the individual atomic orbitals in the polypeptide chain and that the electron dis- tribution may then be more adequately represented by the appropriate molecular orbitals. Delocalization of 7~ electrons in this way has been recognized for simple molecules 3 9 4 but the theory 2 is not completely conclusive since it makes use of methods which may be unsuitable at large internuclear distances. Since the present discussion relies considerably on these calculations, it is worthwhile examining the more recent evidence which appears to justify the conclusions in a more unambiguous way than that originally cited.Electron mobility can be induced in a protein, according to this model, by the absorption of photons of characteristic energy hv greater than the calculated gap between the valence and conduction bands; for the most reasonable stereo- chemical model of the peptide bond, this amounts to some 3 eV. The ex- perimental evidence for such electronic transitions in proteins is somewhat conflicting 5 9 6 although the studies of excited states in tryptophan 7 and tryptophan- containing proteins 8 are of particular interest. Steele and Szent-Gyorgi 8 found that irradiation of a number of proteins in the main absorption band around 280 mp elicited fluorescent and phosphorescent emission at 340 mp and 450 mp.These were respectively interpreted as resulting through transitions from the lowest excited singlet and triplet states to the singlet ground-state of the tryptophan residues. Phosphorescent emission at 450 mp, although a shorter lifetime, could also be elicited by irradiation at about 340mp, a region where the amino acids are transparent ; the emission was, however, reproduced in tryptophan-glucose solutions if the concentration of tryptophan exceeded some 5.10-3 M. It was concluded 7 that tryptophan molecules interact at high concentrations and that this interaction produced a new absorption band at approximately 360 rnp. Apart from possible differences in the nature of the intermolecular forces in the " aggregate ", the observations are entirely similar to those of Scheibe on pseudo- isocyanine 9 where " .. . in the process of polymerization . . ., there occurs a fusion of certain electrons into a new system, causing the new (absorption) band ". The existence of non-localized orbitals extending throughout the protein molecule E 129130 CHARGE TRANSFER PROCESSES IN BIOLOGICAL SYSTEMS is even more clearly expressed by the observed semiconductivity of a number of proteins,lo3 11 the results of which are consistent with a banded structure having an energy gap of 2-3 eV. The Evans-Gergely model would therefore appear to be reasonable in so far as it gives a quantitative account of these critical experiments, but we should be prepared to accept that some modifications to the calculated energy-level scheme will follow a more sophisticated treatment of the problem.EXCITED STATES IN PROTEINS The role which long-lived excited states may play in biological reaction mechan- isms has been discussed by Szent-Gyorgi 12 and the studies of phosphorescence in proteins 8 are of considerable importance in showing that triplet states of these systems can be excited under suitable conditions. Leaving aside for the moment the question of whether the primary photon interaction is with the amino acid residues or with the banded system, we can suppose that a triplet state represents an essential reaction intermediate in the process of denaturation, or loss of specifi- city, induced in proteins by radiation.Of particular interest in studies of the biological effects of radiation has been the finding that a large variety of tissues show a considerable increase in radiosensitivity in the presence of oxygen. More recently, it has been shown 13-15, that nitric oxide is capable of conferring a level of radiosensitivity, comparable with that of an equal concentration of dis- solved oxygen, on a number of cell types. These sensitization effects can now be thought of as being due to the probable enhancement of formation of excited triplet states in proteins. The modification of the probability of single-triplet transitions, in the presence of oxygen and nitric oxide, has been examined by Evans 16917 who largely attributed the effect to a spin-orbit perturbation of the tripIet levels. Their large effect relative to the paramagnetic rare earth ions suggests, however, that their ability to act as electron acceptors in charge transfer com- plexes also contributes to the process.The more usual description of the mechan- ism by which these gases may affect the rate of in vivo reactions also requires the formation of such complexes but as intermediates in the formation of free radicals which are themselves assumed to be responsible for the denaturation. In the case where the excitation is confined initially to an amino acid residue, energy transfer through the molecule could take place by such a mechanism as that of sensitized fluorescence, in much the same way as has been imagined for the photo-decomposition of carbon monoxide from a myoglobin-carbonyl complex.18 But the implications of such excitations on structure are not so obvious as for the situation in which we imagine a valence electron to be excited into the conduction band; the excited triplet state configuration may now be described by the enol structure which can be regarded as arising through a rearrangement of the 7r electrons in the polypeptide chain, RCH I I I I I 1 11 I I I I 1 Franck and Livingston 19 have similarly postulated that, for large dye molecules, the state to which electronic internal conversion takes place may belong to a tautomer of the original molecule while Schmidt 20 considered that the fact that radiation of energy kv - 3-4 eV is needed to induce cancer, suggested that keto- enol tautomerism in proteins represents an essential stage in carcinogenesis.RCH H-N H-N C=O +=F C-0-H RC RCH N-H N-HR. MASON 131 Whether the triplet state can play an important part in determining the biolo- gical effects of radiation could ultimately depend upon the rate of its deactivation and its equilibrium concentration. Ebert et ~21.217 22 demonstrated that a number of gases could reduce the oxygen-dependent radiosensitivity of the broad bean and of mouse Ehrlich ascites tumour cells in the presence of oxygen. The relative effects of these gases were found to be parallelled by their lipid-water partition coefficients so that it was suggested that they owed their properties to the displace- ment of oxygen associated with lipid material in the cell. In fact, for the inert gases, with the exception of argon, the biological effects reflect quite closely the total collision cross-sections of these atoms for slow electrons ; 23 for hydrogen, nitrogen and nitrous oxide, their desensitizing properties are matched by the ob- served average fractional energy gained per collision of a gas molecule with such electrons.24 The correlation is possibly fortuitous in so far as the collisional data appropriate to slow electrons in gases may not closely approximate those for a condensed system. An examination of the in vivo effects of other known quenchers of the triplet state 25926 would be useful in deciding the relative merits of these explanations. Related to the previous discussion is the problem of the mechanism through which a number of biologically active hydrocarbons and their derivatives act.It has been shown 279 28 that protein binding of a number of structurally dissimilar molecules is of causal significance in their induction of cancer and a number of theories relating the carcinogenicity of these molecules to their chemical reactivity have been put forward.29~ 30 Underlying these theories is the assumption that the interaction of the carcinogen with the cellular receiver takes place at or through the reactive K region of the molecule and that the site of reaction is probably some suitable clectrophilic centre in a protein. The conditions for charge transfer in, say, a hydrocarbon-protein complex, have already been indicated ; 2 owing to the narrowness of the bands, the energy levels in the two molecules must be closely matched.In particular, if charge transfer is to take place from the valence band of the protein to an unfilled orbital of the hydrocarbon, the matching criterion would require the hydrocarbon to possess an electronic structure such that the energy difference between a filled and unfilled orbital falls within the range defined by the limits of the two highest filled bands in the protein, that is Ei,r m 3.2 rt 0.2 eV. EjJ is now the energy difference between the ith filled orbital and the jth unfilled level. We have, of course, taken no account of the density function of the bands so that it is to be expected that matching will probably need to be even more critical. This relation provides a surprisingly complete account of the specificity of reaction between aromatic hydrocarbons and proteins.31~ 32 Thus with the simplc Hiickel approximation for the alternant hydrocarbons, the criterion, El, 2 = 3.24 f 0.11 eV, is sufficient to select the six biologically active molecules from among some forty hydrocarbons ; only one inactive molecule, anthanthrene, falls within this range and this may be due as much to inadequate experimental assessments of its activity as to the obvious limitations in the theory.What would appear to be important is that mobility in the hitherto completely filled band of the protein is induced through electron transfer to the second lowest unfilled level in the hydrocarbon, since the energy of the exciton or photon produced on subsequent transitions to the first unfilled orbital correlates quite well with the relative potency of these molecules (table 1).The structural derangements which may occur in the protein following electron transfer in this way are quite likely to be the same as those discussed earlier but are probably insufficient to explain the loss of specific properties by the cell during the carcinogenic process. They may indeed only imply that the restrictions on132 CHARGE TRANSFER PROCESSES IN BIOLOGICAL SYSTEMS the folding of the polypeptide chain, introduced by enolization, lead to a new protein configuration which is unable to fulfil its protective role in a nucleo- protein. Exciton transfer by the protein lattice with subsequent absorption by some suitable " trap " in the nucleic acid could now result in a modified arrange- ment, capable of transmitting new information.The energies of table 1 suggest that alterations in the nucleic acid configuration are effected through hydrogen- bond breakdown, which would undoubtedly affect the molecular specificity. This quenching of excitons by the nucleic acid could, of course, be interfered with by some suitable molecular system: 6-mercaptopurine, one of the more useful chemotherapeutic agents, completely quenches the phosphorescence of riboflavine in a 6 x 10-5 M concentration.12 These anticancer agents may, however, owe their activity to the alternative process of " lone-pair " electron donation to the unfilled band of the protein. TABLE 1 .-ENERGY LEVEL DIFFERENCES FOR BIOLOGICALLY ACTIVE HYDROCARBONS calc.Ezi' (eV) according to scheme mation (a) Huckel (b) Wheland approxi- activity molecule 0.33 + 3 : 4-benzophenanthrene - 1 : 2-benzanthracene 0.75 0.90 + + 1 : 2 : 7 : 8-dibenzanthracene 0.36 - 1 : 2 : 5 : 6-dibenzanthracene 0.60 0.72 ++ 3 : 4-benzpyrene 1.22 1-48 ++++ 3 : 4 : 8 : 9-dibenzpyrene 1 -49 1-55 ++++ The T complex, with which we have described the nature of the hydrocarbon- protein interaction, must, however, only represent a transition state in the re- action path since the protein binding of the hydrocarbons and azo dyes is of a much more definite kind than would otherwise be suggested.27.28 The formation of a carcinogen-protein covalent bond can be thought of as likely to take place at a peptide linkage and as having preceeded via the T complex and some more stable reaction intermediate such as a (T complex.The usual theories of chemical reactivity of hydrocarbons would predict that binding proceeds at the meso- phenanthrene region of those molecules but such binding is not, in itself, to be regarded as important. It is quite possible that protein binding could proceed via some alternative mechanism which did not involve charge transfer from the valence band of the protein and which therefore did not necessarily imply signifi- cant protein derangements. Woodhouse 33 has indeed claimed that proteins can bind a number of inactive hydrocarbons to an extent comparable with their more active analogues: the exact nature of the binding of these molecules is not clear but one must suppose that they do not interfere with the hydrogen bonding of the proteins.SOME GENERAL REMARKS It is clear that the present discussion has involved a considerable number of simplifications, the validity of which will determine the usefulness of this approach. For example, the assumption that a hydrocarbon may act as an electron acceptor in a protein complex is one which is only justified by the results; the difficulties in the argument are related to the fact that for charge transfer to take place, the binding energy of the complex must roughly offset the difference between the ionization potential of the donor and the electron affinity of the acceptor. We have no means of discussing whether this is a probable result but it may be that the relation of hydrocarbon and protein is rather more like that of impurity and host in impurity semiconduction.That is, the hydrocarbon enters the protein lattice but can do so only as a negative ion. In favour of the present theory, however, is a good deal of circumstantial evidence, of which the fact that it en- visages a common mechanism for a wide range of agents may be the most important.R. MASON 133 It is interesting to note that irreversible binding of chromium complexes by the peptide bonds of collagen has been recently demonstrated,w since compounds of the transition elements represent a considerable proportion of the relatively few inorganic carcinogens known. Such protein binding must certainly depend on the electronegativity of these metals vis ci vis the oxygen or nitrogen of the peptide bond.Finally, it should be remembered that the conversion of a number of active molecules into metabolic derivatives constitutes an essential step in their reaction mechanism ; without a detailed knowledge of the nature of these metabolites, which must be regarded as the " true " agents, theories of biological activity in terms of electron distribution and so on will be at best useless and at worst misleading. These investigations are supported by grants from the British Empire Cancer Campaign. NOTE ADDED IN PROOF.-An application of simple charge transfer theory by Tuck 35 shows that the interaction integrals for the adsorption of the inert gases on a constant adsorbate depend on the ionization potential and the polarizability of the gas concerned. For charcoal the interaction integrals range from 0.64 eV in the case of helium to 1.55 eV for xenon ; one would expect the absolute values of these integrals to be quite different for protein adsorption but their relative values again reflect the desensitizing properties of these gases.Such a model may prove to be more acceptable than that originally suggested, viz. that these gases owe their desensitizing properties to their collisional deactivation of excited states in proteins. 1 Szent-Gyorgi, Nature, 1941, 148, 157. 2 Evans and Gergely, Biochim. Biophys. Acta, 1949, 3, 188. 3 Gupta, Proc. Physic. SOC. A , 1954,67,643. 4 Mason, Acta Cryst., 1956,9, 405. 5 Beaven, Holiday and Jope, Faraday SOC. Discussions, 1950,9,406. 6 Schauenstein, Faraday SOC. Discussions, 1950,9, 491. 7 Isenberg and Szent-Gyorgi, Proc.Nat. Acad. Sci., 1958, 44, 519. 8 Steele and Szent-Gyorgi, Proc. Nat. Acad. Sci., 1958, 44, 540. 9 Scheibe, Kolloid-Z., 1938, 82, 1. 10 Eley, Parfitt, Perry and Taysum, Trans. Faraday Soc., 1953,49, 79. 11 Eley, Faraday SOC. Discussions, 1955,20, 273. 12 Szent-Gyorgi, Bioenergetics (Academic Press, New York, 1957). 13 Howard-Flanders, Nature, 1957, 180, 1191. 14 Kihlam, Expt. Cell. Res., 1958, 14, 639. 15 Gray, Green and Hawes, Nature, 1958, 182, 952. 16 Evans, J. Chem. SOC., 1957, 1351. 18 Franck and Livingston, Rev. Mod. Physics, 1949, 21, 505. 19 Franck and Livingston, J. Chem. Physics, 1941, 9, 184. 20 Schmidt, Naturwiss., 1941, 29, 146. 21 Ebert, Hornsey and Howard, Nature, 1958, 181,616. 22 Ebert and Hornsey, Nature, 1958, 182, 1240. 23 Massey and Burhop, Electronic and Ionic Impact Phenomena (Clarendon Press, 24 Healey and Reed, The Behaviour of Slow Electrons in Gases (Amalgamated Wireless, 25 Terenin and Ermolaev, Trans. Faraday SOC., 1956, 52, 1042. 26 Ermolaev and Terenin, J. Chim. Physique, 1958, 55, 698. 27 Miller and Miller, Cancer Res., 1952, 12, 547. 28 Heidelberger and Moldenhauer, Cancer Res., 1956, 16,442. 29 Pullman and Pullman, Canctrisation par les substances chimiques et structure molec- 30 Pullman and Pullman, Advances in Cancer Research (Academic Press, New Yo&, 31 Mason, Nature, 1958, 181, 820. 33 Woodhouse, Brit. J. Cancer, 1955, 9, 418. 34 Gustavson, Nature, 1958, 182, 1125. 17Evans, J. Chem. SOC., 1957, 3885. Oxford, 1952). Australasia, Sydney, 1941). ulaire (Masson, Paris, 1955). 1955), vol. 3. 32 Mason, Brit. J. Cancer, 1958, 12, 469. 35 Tuck, J. Chem. Physics, 1958, 29, 1108.

ISSN:0366-9033    

DOI:10.1039/DF9592700129

出版商:RSC    

年代:1959

数据来源: RSC

摘要:

ELECTRONIC ENERGY TRANSFER IN HAEM PROTElNS BY G. WEBER AND F. J. W. DALE Dept. of Biochemistry, Sheffield University Received 16th February, 1959 The fluorescence efficiency of the tryptophan fluorescence of haem proteins is very small, in most cases smaller than 0.002, while after removal of the haem is around 0.2. The fluorescence efficiency of crystallizable conjugates of the haem proteins with di- methylamino naphthalene sulphonyl chloride (DNS) before and after removal of the haem has been determined. On the assumption of the uniform random distribution of the labels over the spherical protein surface, the one-haem proteins myoglobin and horse-radish peroxidase give as the distance R at which the probability of transfer of the excited state to the haem equals the probability of emission by the DNS residue, a value of 42 A, to be compared with 58A calculated by the theory of Forster.In the four-haem proteins haemoglobin and catalase the values of R are 65 8, and 66 8, if the haem sare supposed to be crowding together at one place on the surface of the molecule and 47 8, and 49 8, respectively if two pairs of haems are assumed to occupy two diametrically opposite places on the surface. The values quoted are for the molecules with haem in the ferric valency state. Reduction to the ferrous state is accompanied by an increase in the quenching efficiency of 1.5 to 2 in the three proteins thus studied, in good quantitative agreement with the change of 1.7 in the computed overlap integral of the absorption and emission spectra of the conjugates.The photodecomposition of the carbon monoxide compounds of several haem proteins has shown that light absorbed by the aromatic amino acid residues is active in promoting the photochemical changes.1 A pre-requisite of these changes is the transfer of the electronic energy from the oscillator originally excited to the haem. This transfer can be investigated by a study of the yield and lifetime of the fluorescence of the intact haem protein in comparison with the corresponding quantities observed when the haem is split off. The con- ditions necessary for this transfer to take place are apparently present in the haem proteins : the fluorescence emission from the aromatic amino acids 2 is well covered by the absorption band of the haem and the average distance among random points in the protein molecule is of the order at which transfer may be expected to occur (1-3 mp).Electronic energy transfer to the haem can also be studied in the fluorescent naphthylamido conjugates of the haem proteins,3 where the intensity of the fluorescent emission in the intact and haem-free protein conjugate is capable of yielding similar information. THEORmCAL If the ith fluorescent molecule is placed at distance rv from the jth quenching centre the ratio of the fluorescence intensity observed to that observed in the absence of quencher is : where A = transition probability of emission and v transition probability of transfer of the singlet state from i toj. For this van der Waals type of transfer it is known 4 134G . WEBER AND F.J. W. TEALE 135 that v/A = R6/r$, ru being the actual distance from i to j and R a characteristic distance for which v = A. The fluorescence intensity from all i centres is i With ni equal fluorescent centres and n2 equal quenching centres, we have 1 1 + 2 (R6it-5) * n2 - F = Fo 1 (3) In the transfer of the excited state from the aromatic residues of the protein to the haem, any calculations of the value of R from the experimental data require the detailed knowledge of the r~ values. While advance in the knowledge of the structure of the proteins concerned may make this calculation possible in the future, all that can be said at present is that one may expect a rough correlation between protein size and fluorescence efficiency to exist. More interesting information can be obtained in the study of the conjugates of the proteins with dimethylamino naphthalene sulphonyl chloride (DNS conjug- ates).This reagent combines with the amino groups, particularly the €-amino groups of lysine 3 and if these are distributed rather uniformly over the surface of the protein and possess similar reactivity, one may expect to obtain conjugates in which the quencher centres have around them a population of fluorescent centres distributed randomly over the surface of the molecule. If this is assumed spherical for simplicity the value of FIFO can be calculated explicitly as a function of the parameter R/2p, where p is the radius of the sphere. The approximate dimensions of the globular molecules studied are known so that R can be finally evaluated.Replacing the summations in eqn. (3) by the appropriate integrals and introducing the variable z = r/2p, eqn. (3) becomes f ( z ) being the fraction of the molecules at distance r, a function of j , from the quenching centre. For one haem, the last equation gives The lifetime of the excited state of the fluorescence of the quenched system 5 is related to the lifetime of the unquenched one TO by the relation, since the lifetimes are weighted according to intensity. For a one-haem spherical molecule, Eqn. (5) and (7) have been used in calculations of FIFO and ;/TO as a function of R/2p by means of a digital computer, and the values are shown as a graph in fig. 1. For the four-haem proteins, if the haems are supposed to be together at one point136 ENERGY TRANSFER I N HAEM PROTEINS Rl2P FIG.1.-Relative fluorescence e5ciency FIFO and lifetime 7/70 as a function of character istic distance R and radius of sphere p for one quenching centre. 0.9 - 0.8 - 0.7 - 0.6 0.5 0.4 - - FIFO - 0.3 0 . 2 0.1 - - - 0.1 0 . 2 0.3 0 6 0 . 5 0.b 0.7 0.8 0-9 1.0 1.1 1.2 RPP FIG. 2.-Relative fluorescence efficiencies as function of characteristic distance R and radius of sphere for two quenching centres. 2-2, quenching centres of equal weight at opposite ends of diameter. 2-1, quenching centres of weight 2 to 1 at opposite ends of diameter.G . WEBER AND F. J . W. TEALE 137 in the molecule, eqn. (5) and (7) hold but the R constant calculated is R4 = 4*R. Computations have also been made for the case in which the four haems are in two pairs at diametrically opposed ends of the molecule, in which case, F ZdZ - = z 1 1 +(a,"('+ -).1 ZS (1 - 22)3 The results are shown in fig. 2 which also includes calculations of a model like the previous one in which one of the four haems is missing. EXPERIMENTAL MATERIALS AND METHODS PREPARATTON OF THE HAEM PROTEINS.-MyOglObin was extracted from sperm whale muscle by the method of Keilin and Schmid.5 It was recrystallized three times from 0.8 saturated ammonium sulphate at neutrality. Horse-radish peroxidase was a gift from Prof. D. Keilin and was used without further purification. Pig heart cytochrome c was prepared by the method of Margoliasch.6 It was purified by several passages through ion-exchange resin. Haemoglobin was prepared from horse blood erythrocytes by the method of Drabkin 7 and recrystallized three times as the carbon monoxide compound from 0-75 saturated ammonium sulphate at pH 7.5.Catalase were prepared from beef liver by the method of Mosimann 8 and from horse blood erythrocytes by the method of Bonichsen.9 1-dimethylamino naphthalene-5-sulphonyl chloride was carried out by the method of Weber.3 The haem protein concentrations were in the range of 0.2 - 1 x 10-3 M and sulphonyl chloride equivalent to twice the desired conjugated group to protein ratio was added. The total acetone concentration was kept below 5 % by volume. The free sulphonic acid was eliminated by filtration through Dowex 2 resin.10 This treatment was repeated immediately before examination of the fluorescence in order to eliminate a small amount of free sulphonic acid which is formed on storage of the protein as a result of hydrolysis of the sulphonamide. protein-haem link was broken by the method of Drabkin.11 The molarity of the naph- thalene sulphonamido residues was estimated spectrophotometrically in the haem-free solution by the optical density at 340 mp using a molar absorption coefficient 10 of 4.3 x lo3 cmz/rnM.The total protein in solution was then determined by dry weight. For cytochrome c where the haem could not be separated, the amount of naphthalene residues was only estimated from the amount of sulphonyl chloride used, assuming a 60 % efficiency of coupling. PURITY OF THE CONJUGATES-The high efficiency of the quenching of the fluorescence in all the haem proteins studied made it imperative to exclude any haem-free molecules present before or produced during the conjugation procedure.The need for purity can be judged by the fact that 2 % of haem-free haemoglobin is sufficient to double the apparent quantum efficiency of the solution. Haem losses during conjugation could be minimized by keeping the haem in the reduced form. Absence of haem-free protein was further insured by crystallization of the actual conjugates from neutral ammonium sulphate solutions of 0.7-0.9 saturation. Several fractions of crystals were obtained, the crystals of conjugates with a higher number of labelling groups appearing first. In con- trast to the crystallization of the haem proteins the crystallization of the conjugates was very slow and the crystals appeared as thin sheaves of needles instead of large tabular plates.Cytochrome c and horse-radish peroxidase, in which the haem is not lost, were used after conjugation without further purification. All the catalase specimens pre- pared contained haem-free protein, which was removed after conjugation by chromato- graphy on a paper powder column with 0.4 saturated ammonium sulphate at pH 5. The separation of a strongly fluorescent band was observed in ultra-violet light, and the relatively non-fluorescent brown band was retained, PREPARATION OF FLUORESCENT CONJUGATES.-ReaCtiOn Of the haem proteins with DETERMINATION OF THE NAPHTHALENE RESIDUE TO PROTEIN MOLECULAR RAno.-The138 ENERGY TRANSFER IN HAEM PROTEINS FLUORESCENCE mAsmEmNm.-The fluorescence from the intact haem proteins and the conjugates was determined in solutions of equal molarity in the 10-6 M range.At this concentration the absorption of both the exciting and the fluorescent light was negligible. Hg 366 mp light was used for the excitation of the conjugates and 280 mp light obtained by means of a xenon arc and grating monochromator for the excitation of the fluorescence of the intact haem proteins. The fluorescence was measured in both cases with photomultiplier (E.M.I. 6255) and galvanometer. Separation of the haem from the conjugate was accomplished by the method of Drabkin,ll and also by that of Teale,l2 in which the haem is extracted from the acid protein by methyl-ethyl ketone. DISCUSSION The results are summarized in table 1. Concerning the ultra-violet fluorescence of the haem proteins it is evident that quenching by transfer of the excited state to the haem takes place practically completely with the exception of horse-radish peroxidase in which fluorescence of the order of 15 % of that of the split globin is present.The absolute quantum yields of fluorescence of the haem-free globins are in the region of 0.2 so that the radiationless transitions from the singlet excited state are about 5 times more probable than the fluorescence. On the other hand, according to tab'e 1 the transfer of the excited state to the haem has a probability at least 100 times greater than the fluorescence, and therefore some 20 times TABLE 1 Fo/F is the Ratio of the Fluorescence Efficiency Observed after Removal of the Haem, to that Measured in the Intact Protein Ultra-violet fluorescence of native haem proteins All the observations refer to proteins in the Fe3+ state.haemoglo bin catalase horse-radish protein myoglobin peroxidase FolF > 100 7 > 100 > 100 Visible fluorescence of dimethylamino naphthalene sulphonamido conjugates of haem proteins naphthalene protein residues/molecule 'dF myoglobin Fe*+ myoglobin Fe3+ horse-radish Y, Y, peroxidase Fez+ 9 ) horse-radish peroxidase Fe3 + Y 9 cytochrome c (Fez+, Fe3+) haemogIobin Fe2+ haemoglobin Fe3+ catalase Fe3 + 9 ) > Y Y, 1 3-4 1 3-4 1 3-4 1 3-4 1-4 1 3-4 1 3-4 1 3-4 32 18 15 9 11 6 7 3.5 > 100 R4 60 67 54 67 40 65 38 65 7 69 5 64 NA) 47 43 41 37 53 46 48 41 > 30 R2. 2 51 50 47 47 52 45 & is the value calculated for the four haems together (fig.1); R2, 2 is calculated for the haems in pairs (fig. 2). The diameter of the haem proteins used in the calculations in 8, are : myoglobin 34 ; haemoglobin 5 4 5 ; horse-radish peroxidase 47 ; cytochrome c 16.5 ; catalase 84.a. WEBER AND F. J . W. TEALE 139 greater than the other competing radiationless processes. On these figures it appears that the quantum yield of the indirect photochemical effects resulting from the transfer of radiation originally absorbed by the aromatic amino acids to the haem can have a quantum yield as high as that resulting from the direct excitation of the haem, as has indeed been observed.13 In horse radish peroxidase in which the quantum yield of the fluorescence is of the same order as the ratio of fluor- escence to transfer, it may be expected that the indirect excitation will be only half as effective in promoting photochemical changes as the direct excitation of the haem.Any conclusions as regards the quenching effects of the haem on the sulphon- amido conjugates depends on the effects that would be introduced by part of the labelling molecules occupying a definite place in the protein rather than being distributed at random. Such effects can be estimated by considering that a frac- tion 1-x of the labels is distributed randomly over the surface and a fraction x Z = r/2p FIG. 3.-Effect of non-random labelling on quenching by transfer. Abscissa = z = r/2p ; ratio of actual distance of non-random fraction to diameter of sphere. Ordinate= signed fluorescence efficiency difference from the random case ; x = non-random fraction.The three curves are for R/2p = 0.7, 0.9, and 1.3 respectively. occupies a definite place at distance z = r/2p from the haem. Two variables have to be considered: z and x together with the parameter R/2p. Fig. 3 shows the values of the difference between Ill0 observable in each case and (l/Zo) random, the value shown in fig. 1, as a fraction of the latter, plotted against z for the range of values of R/2p which are of interest in our case, namely 0.7-1-3. The deviation from the random case is found by multiplying the ordinate by x, the fraction non-randomly distributed. Very considerable departures from the random dis- tribution are required to make a difference in the calculated values of R.If the random and non-random fractions are equal a maximum error of - 100 % to + 200 % in the value of I/Zo may be found but this results in only + 10 % and - 20 % errors respectively in the calculated R. Experimentally the variation of quenching ratio with degree of labelling gives a means of detecting departures from uniformity of labelling over the surface. As the number of fluorescent residues per molecule is increased the distribution should become more regular and the values of R calculated more likely to be correct. The increase in the degree of labelling in myoglobin and horse-radish peroxidase produced some140 ENERGY TRANSFER IN HAEM PROTEINS changes in R as shown in the table. The four values of R give an average of 42 A for the ferric proteins and 47 A for the ferrous proteins. The heavily labelled samples have systematically higher values of 1/10 than those labelled with one DNS group, indicating that the departure from the random distribution is in the direction of an accumulation of DNS residues nearer to the haem.DISPOSITION OF THE HAEMS IN THB FOUR-HAEM PROTEINS In the four-haem proteins, haemoglobin and catalase, the values of R cal- culated on the assumption of all the haems being together at one place in the molecule are respectively 65 and 68A. R4 = 47A requires the non-random kK FIG. 4.-Absorption spectra of ferromyoglobin (Fe2+M) and ferrimyoglobin (Fe3+M) and fluorescence spectra of conjugate (dashed line). The latter is plotted in with a maximum of 4.3 x 103 (molar absorption of corresponding absorption band).The absorption spectra are only represented for v > 16,000 cm-1. fraction to be over 90 %, which is clearly inconsistent with the very small change in quenching efficiency with degree of labelling observed, and shown in table 1. Jf, on the other hand, the model of four haems in opposite pairs (eqn. (8) and fig. 2) is used in the calculations it is found that R is 47A for haemoglobin and 48A for catalase, values which show much better agreement with those calculated for the one-haem proteins. EFFECT OF THE VALENCY STATE OF THE IRON The valeiicy change of the iron in the haem proteins produces changes in the absorption spectrum. Fig. 4 depicts the changes in myoglobin. On reductionG. WEBER AND F. J . W. TEALE 141 an appreciable change 14 in the overlap integral J of the absorption and emission spectra takes place.The values of J obtained by graphical integration are : ferromyoglobin 2.8 1010 cm3 mM-2, ferrimyoglobin 1.7 1010 cm3 mM-2. The ratio of the two J values is 1.65. The six pairs of values quoted in table 1 give an average for the quenching ratio of reduced to oxidized protein of 1-74 f0.24. The agreement with Forster’s theory which predicts proportionality between overlap integral and probability of transfer may be considered satisfactory. This same theory predicts a value for R given by R6 = 0.66 x 10-33 ( ~ o J / v $ ) cm6, in which we have assumed the refractive index to be that of the protein: 1.5. With vo = 20,OOOcm-1, and TO = 1.3 x 10-ssec as experimentally determined by Steiner and McAlister 15 for other DNS conjugates, R = 58 8, for the ferric proteins and 63A for the ferrous proteins.These values are some 40 % larger than those estimated from the experimental data, and correspond to quenching efficiencies different from the observed ones by a factor of 10, which is apparently beyond the systematic errors involved. This disagreement is not surprising when it is considered that the theory predicts values of 50, 55 and 808, for the characteristic distance of transfer among molecules of fluorescein, rhodamin B16 and chlorophyll A, while the observed values are 26, 30 and 368, respectively. The financial help of the Medical Research Council and the Medical Research Fund of Sheffield University is gratefully acknowledged. Part of this work was carried out during the tenure of a fellowship of the Medical Research Council by one of us (F. J. W. T.). We thank Prof. D. Keilin for a gift of horse-radish peroxidase and Mr. P. H. Blundell of the Department of Applied Mathematics of Sheffield University for carrying out the computations. 1 Biicher and Kaspers, Naturwiss., 1946, 33, 93. Bucher, Adv. Enzymol., 1953, 14,l. 2 Teale and Weber, Biochem. J., 1957, 65, 476. 3 Weber, Biocheni. J., 1952, 51, 155. 4 Perrin, Ann. Physique, 1932, 17, 283. 5 Keilin, J., and Schmid, Nature, 1948,162, 496. 6 Margoliasch, Biochem. J., 1954, 56, 535. 7 Drabkin, Amer. J. Med. Sci., 1945, 209, 268. 8 Mosimann, Arch. Biochem., 1951,33,487. 9 Bonichsen, Arch. Biochem., 1947, 12, 83. 10 Weber, Faraday Soc. Discussions, 1953, 13, 34. 11 Drabkin, J. Biol. Chem., 1945, 158, 721. 12 Teale, Biochim. Biophys. Acta, 1959, 35, 543. 13 Bucher and Kaspers, Biochim. Biophys. Acta, 1947, 1,21. 14 Forster, Ann. Physik, 1947, 2, 55. 15 Steiner and McAlister, J . Polymer. Sci., 1957, 24, 105. 16 Weber, Trans. Faraday Soc., 1954, 50, 552.

ISSN:0366-9033    

DOI:10.1039/DF9592700134

出版商:RSC    

年代:1959

数据来源: RSC

摘要:

MODIFIED REACTIVITY OF HAEMOGLOBIN FOLLOWING LIGHT ABSORPTION BY Q. GIBSON Sheffield University Received 16th January, 1959 When carboxyhaemoglobin is broken down by the action of light, the newly formed reduced haemoglobin has a higher rate of combination with carbon monoxide than ordinary reduced haemoglobin. " Newly-formed " haemoglobin is converted to " ordin- ary " haemoglobin with a rate constant of about 200 sec-1 at pH 9 and 0;O. The rate constant for the combination of "newly-formed" haemoglobin with CO is 1-8 x 106 M-1 sec-1 at 0;O and the activation energy is 5.6 kcal (cf. ordinary haemoglobin 4 x 104 M-1 sec-1 and 10.5 kcal). In 1896 Haldane and Lorrain-Smith 1 found that the results of the carmine titration method for determining the composition of mixtures of oxy- and carboxy- haemoglobin depended on the lighting of the laboratory, and went on to prove that carboxyhaemoglobin (COHb) is photosensitive.It has since been shown by Buecher and Kaspers 2 that the absorption and action spectra of carboxymyo- globin are identical over the range 200-450 mp ; thus energy transfer must take place from the aromatic amino-acids of the protein to the haem group. The quantum yield was 1 throughout, so this transfer is clearly an efficient process. It has been known for many years that light of wavelengths shorter than 300 mp will produce major irreversible changes in proteins, and haemoglobin is among those attacked. With visible or near ultra-violet light, which is absorbed chiefly by the haem group, it has been supposed, however, that the dissociation COHb +- CO + Hb is the only reaction taking place.Experiments described by Gibson 3 suggest that this may not be true, as under some circumstances the immediate product of the photodecomposition of COHb is not ordinary reduced haemo- globin (Hb) but a short-lived quickly-reacting species which will be written Hb*. Several explanations of the observations are possible and will be briefly discussed. Gibson 3 found that, following the photochemical dissociation of COHb by light of wavelengths longer than 310 mp, there was present in the solutions a pig- ment whose spectrum in the Soret region was closely similar to, but not identical with, that of reduced haemoglobin. This material Hb* was converted spontan- eously into Hb with a rate constant at 0" and pH 9.1 of 250 sec-1.It reacted with CO to re-form HbCO with a rate constant at 0" of 2 x 106 M-1 sec-1 (cf. the rate constant for the combination of Hb with CO at pH 9.1 and 0" of 5 x 104 M-1 sec-1 given by Gibson and Roughton 4). The energy of activation for Hb* combining with CO was 5.6 kcal at pH 10.6, compared with 10.5 kcal for Hb combining with CO. The proportion of Hb* formed is a function of pH, being least at pH 6-6. It appears to depend also on the rate at which CO is removed from combination by the photolysis flash, though for technical reasons few flash intensities have been examined. These findings may be related to Gibson and Roughton's 4 measurements of the rate of combination of the four successive molecules of CO with Hb. They found that whereas the first three molecules to combine do so at roughly similar rates, the fourth molecule combines about 40 times faster.This difference in behaviour between free and combined Hb is thought to be due to a structural change in the protein rather than to a difference in individual haem molecules; 142Q. GIBSON 143 certainly, saturation is associated with changes in crystal form and solubility, as well as in the isoelectric point. With these results in mind, it seems reasonable to think of Hb* as a haemoglobin molecule which has lost its ligand molecules through photochemical action in a space of time so brief that the reorganization of the protein normally associated with the loss of the second ligand molecule has not had time to occur. In slightly different words, Hb* may be regarded as combined haemoglobin minus its ligand molecules.This qualitative picture will explain satisfactorily the high rate of combination of Hb* with COY which is similar to the rate for the combination of the fourth and last molecule of CO with Hb, i.e. the reaction Hb4(C0)3 + CO +- Hb4(C0)4 : the similarity extends indeed to the ratio between the rates of combination of 0 2 and CO. Thus for Hb* the ratio is 4/1 ; for Hb4(C0)3 Gibson and Roughton 5 found 3.5 : 1. The nature of the change in the protein is not known. It may well involve sulphydryl groups, since the transition Hb* -+ Hb is much slower in the presence of mercurials. St. George and Pauling 6 have suggested that the haem groups may lie in a " crevice " in the protein, and it is naturally tempting to relate the increase in the rate of CO combination on passing from Hb4 to Hb4(C0)3 to the opening up of such a crevice structure.The rates and activation energies for the reactions Hb* + CO and Hb + CO do not support such an idea, for the difference of 5 kcal should correspond to a difference in rate of about 1000-fold, instead of the observed %-fold, while, if steric factors were favourable, a still greater increase might be looked for. In fact, the steric factor is less favourable for the combination of Hb* than for Hb. Finally, it is interesting to consider if the observations should be regarded as an example of energy transfer. A photochemical effect is produced giving rise to Hb*, and this action is produced at a distance since the protein rather than the haem is implicated. On the other hand, unless all the ligand molecules are stripped from the haemoglobin molecule within a brief space of time, Hb* will not appear, as the changes in the protein required to give Hb will take place faster than the removal of ligand. On this picture, the removal of the ligand molecule would be a trigger step followed by a spontaneous change in the protein. Thanks are due to the Medical Research Council for contributions to the cost of the work. 1 Haldane and Lorrain-Smith, J. Physiol., 1896, 20, 497. 2 Buecher and Kaspers, Biochim. Biophys. Acta, 1947, 1, 21. 3 Gibson, Biochem. J., 1959, 71, 293. 4 Gibson and Roughton, Proc. Roy. SOC. B, 1957,146, 206. 5 Gibson and Roughton, Proc. Roy. SOC. By 1955,143,310. 6 St. George and Pauling, Science, 1951,114,629.

ISSN:0366-9033    

DOI:10.1039/DF9592700142

出版商:RSC    

年代:1959

数据来源: RSC

摘要:

THE ROLE OF THE TRIPLET STATE IN REACTIONS SENSITIZED BY CHLOROPHYLL BY ROBERT LIVINGSTON AND A. C. PUGH Division of Physical Chemistry, University of Minnesota Received 28th January, 1959 A possible mechanism of photosynthesis involves an inductive-resonance migration of energy of excitation between chlorophyll molecules leading to the trapping of this energy by a pigment molecule which is so situated that it can initiate the sequence of bio- chemical acts that constitute the secondary mechanism of photosynthesis. It is sometimes assumed that this pigment molecule is complexed with a substrate molecule. In studying, in solution, the quenching of the triplet state of chlorophyll by retinene, it was observed that the rate of disappearance of the triplet approaches a limiting value as the retinene concentration is increased.A possible explanation of this result is that retinene and chlorophyll form a moderately stable addition compound whose triplet state has a half- life about one-sixth of that of simple chlorophyll. The triplet state is an essential intermediate in chlorophyll-sensitized photo- chemical reactions occurring in homogeneous solutions.1 This can be demon- strated by comparing the photochemical yields of these reactions to the quantum yields of fluorescence in the same solutions. It has been directly confirmed by flash-photolytic measurements.2.3 Whether the triplet state plays a similar role or any important part in natural photosynthesis is still an open question. Rosen- berg et aZ.4 were unable to detect the characteristic absorption of the triplet state when they exposed a suspension of chloroplasts to a flash which was capable of converting 90 % of the chloroplyll molecules in a dilute solution into their triplet state.Therefore, if the triplet is an important intermediate in photosynthesis, its mean life in vivo must be much shorter than it is in dilute solutions. This is a surprisingly high value. The maximum fluorescence yield of chlorophyll-a in " wet " 6 organic solvents 7 is 33 %. Combining this value with the empirical equation 8 for the self-quenching of the fluorescence of chlorophyll-a in solution, +j/$f = 1 + 6270 m, we would predict that the fluorescence yield of 0.1 m solution would be 0.5 %-less than one-fifth of that observed for chlorophyll in chlorella.An estimate of the yield in vivo may be obtained by multiplying the maximum yield in vitro by the ratio of the directly measured 9 half-lives of fluorescence in chlorella and in solution. The value obtained in this way is about 10 %, which is consistent neither with occurrence of self-quenching nor with a reasonably efficient yield of photo-synthesis. Rabinowitch and Brody 9 suggested that their result (the ratio of the measured half-lives) is evidence that either three-quarters of the chlorophyll in the chloroplast is in a non-fluorescent state or else that a 10 % yield is characteristic of the cell when it is illuminated with a flash of very short duration and relatively low energy. The latter explanation seems preferable since the former appears to necessitate additional ad hoc hypotheses.For our immediate purpose, the important conclusion to be derived from these data is that the fluorescent state of chlorophyll is not appreciably self-quenched in vivo. The self-quenching of fluorescence in concentrated chlorophyll solutions results 8.10 from the migration of the energy of excitation between pigment molecules until it encounters an adventitious, non-fluorescent dimer. The absence of self-quenching in the chloroplast may be due either to the failure of 144 In intact cells, the quantum efficiency of fluorescence 5 is about 2.7 %.R. LIVINGSTON AND A . C . PUGH 145 the (inductive-resonance) mechanism of energy transfer or to the lack of suitable quenching dimers. However, the degree of depolarization of fluorescence is about the same in the chloroplast 11 as it is in 0.1 rn solutions in viscous solvents.*a This suggests that excitation migration occurs with normal efficiency in the chloro- plast.If the chlorophyll molecules in the grana are arranged in unimolecular films, it is possible that their edge-to-edge contacts are not sufficient to render them non-fluorescent. If we accept the view that there is no self-quenching in the chloroplast and that the only important energy sinks are those which lead to the photosynthetic chemical reactions, a 3 % yield of fluorescence appears to be compatible with a high efficiency of photosynthesis. Let us assume that the only important reactions of the chlorophyll molecules in their fluorescent state are the following. GH* --f GH + hvf fluorescence, (1) GH* +--f GH internal conversion, (2) ( 3 ) In terms of this simple scheme, the fluorscence and primary chemical yields can be written as, R + GH* ++ GH + P initiation of the chemical chain.and ki + k2 + F3) $them. = where 273 is a function of the “ concentration ” or availability of the substance which acts as the energy sink. Since in “ wet ” solvents 6 the maximum yield of fluorescence is 0-3, k2 must be twice as great as kl. If we assume that the ratio kZ/kl is the same in the chloroplast as it is in wet solvents, it follows that, k1/(3kl + F3) = 0.027 and F3 = 33kl. The corresponding value for the yield of the primary act of photosynthesis is $=hem. = 33/36 = 0-92, which is compatible with the known high efficiency of the overall reaction.It is also possible that step 2 produces triplet rather than ground-state molecules in the chloroplast, just as it does in solution.1 If the triplet molecules, so formed, contribute efficiently to the initiation of the chemical steps, the value of +them. should be increased to 0.97. If we postulate that all of the initially excited chlorophyll molecules are con- verted into their ground triplet states before initiating the sequence of biochemical reactions, we must conclude that (in vivo) the ratio, k2/kl, is 33, rather than 2. A further consequence of this postulate is that the self-quenching of the triplet state, GH + GH’ --f 2GH, must be much less efficient in the chloroplast than it is in non-viscous solutions. In pyridine 12 the rate constant for this bimolecular reaction is 3 x 107 rn-1 sec-1, which extrapolates to a half life of 2 x 10-7 sec for the triplet state in a 0.1 rn solution.This is probably too short to allow efficient competition from either a diffusional, bimolecular reaction or the migration 13 of the energy of excitation to a chlorophyll molecule which is already complexed with a suitable reactant. However, unlike the self-quenching of fluorescence, the self-quenching of the triplet state appears to be a diffusional, viscosity-dependent reaction.14 It would not be surprising, therefore, if this process were negligible in viuo. Another and more serious objection to this mechanism is that it leads to the prediction that a flash of moderate intensity should produce readily detectable concentrations of the triplet state in chloroplast suspensions.4 A plausible modification of the first mechanism may be stated as follows.The energy of excitation (of the fluorescent state) migrates to a chlorophyll molecule which is in a specially reactive situation, and it then undergoes a transi- tion from its fluorescent to its (relatively long-lived) triplet state. This favourably located, triplet-state molecule starts the chain of biochemical reactions. As a146 ROLE OF TRIPLET STATE result, the mean life of the triplet state molecules will be markedly shortened in an actively photosynthesizing cell. An elaborated variation of this mechanism was proposed 15 by James Franck in explanation of the detailed results of kinetic and physical studies of photosynthesis.While it is far from proven that the primary act of photosynthesis corresponds to the mechanism described above, it is at least plausible that the sequence of biochemical reactions is initiated by a chlorophyll molecule in its triplet state. Accordingly, it seems worthwhile to investigate the chemical reactions of such molecules. This can be done by measuring the effects of various added compounds upon the rate of decay of the triplet state of chlorophyll in fluid solutions. Fujimori and Livingston 3 have published the results of a few such measure- ments. They found that the oxidizing agents, quinone and oxygen, strongly quench the chlorophyll triplet, although the corresponding irreversible reactions are negligibly slow. The reducing agents, ascorbic acid, allylthiourea and phenyl hydrazine, were without effect, even at concentrations as high as 0.1 in.Phenyl hydrazine was tested in methanol ; allylthiourea and ascorbic acid, in pyridine. The rate constants for the bimolecular reactions (e.g. 0 2 + GH’ -+ GH + 0 2 ) of quinone and oxygen were 2.4 x 109 and 1.1 x 109 m-1 sec-1 respectively. P- carotene and some of the carotenoids also proved to be efficient quenchers, having quenching constants in the range from 1.5 x 109 to 7 x 108 m-1 sec-1. Recently, Dr. S . Ichimura, working in this laboratory, has confirmed the quenching action of p-carotene and, using toluene as the solvent obtained a value of 6.5 x 108 rn-1 sec-1 for this constant. He also observed that rn-dinitrobeniene quenched the triplet state of chlorophyll in toluene and found kQ = 1-2 x 109 m-1 sec-1.This latter result is in contradiction to the claim of the previous workers 3 that rn-dinitrobenzene is a non-quencher. The earlier negative results were based upon only a few experiments and may have been in error. Since the quenching action of the carotenes was unexpected, we have investigated the effect of an analogous compound, retinene. A purified sample of all-trans retinene, which was very kindly furnished by Dr. R. Hubbard of the Harvard Biological Laboratories, was used in these experiments. To prevent its photo- chemical isomerization,l6 the cuvette was jacketted with a solution of potassium dichromate, which transmitted light absorbed by chlorophyll only. Preliminary measurements indicated that retinene, like carotene, quenches the triplet of chlorophyll, with a bimolecular rate constant of about 3 x 108 m-1 sec-1.How- ever, a more careful analysis of the data shows that the rate of decay of the triplet is not a linear function of the retinene concentration but at high concentrations appears to be approaching a limiting value, some five- or six-fold greater than the unquenched rate. This result is not consistent with the commonly-assumed bimolecular mechanism of quenching. One possible interpretation of this result is that, in solution, retinene, R, and chlorophyll are in equilibrium with an addition compound, R + GH = R*GH and that the half-life of the triplet state of the addition compound is about six- fold less than that of simple chlorophyll. Since (experimentally) the red end of the absorption spectrum of chlorophyll and the initial yield of the triplet state are unaffected by the presence of retinene, it follows that the ratio of the initial con- centrations of the triplets of simple and retinene-complexed chlorophyll is equal to the ratio of the equilibrium concentrations of these species in their ground states.Introducing the symbols g and r for the stoichiometric concentrations of chlorophyll and retinene and x for the equilibrium concentration of the addition compound, X K = x2 - ( g + r + l/K)x + rg = 0. (g - x)(r - 4’ or147 Since g < r, for all solutions studied, x < 3(g + r 4- l / K ) , we may make the simplifying approximation that R . LIVINGSTON AND A. C. PUGH For the chlorophyll concentrations and flash intensities used, the absolute rate of the second-order decay, 2GH’ -+ 2GH, is small compared to the first-order process, GH’ +.GH, we may neglect the second-order process, and write for the initial rate of disappear- ance of the triplet in the presence of retinene, FIG. 1.-The initial values of the relative rates of disappearance of the triplet state of chlorophyll in solutions containing various concentrations of rethene. Introducing the proportionality factor, we may write In the absence of retinene Therefore, - (d[GH’]/dt)o UO = klatg.148 ROLE OF TRIPLET STATE In fig. 1, the experimental values of the ratios of the initial rates (with and without retinene but at the same total chlorophyll concentration) are plotted, as small circles, against the stoichiometric concentration of retinene.The vertical lines indicate the apparent standard deviations of these ratios. The solid-line curve is a plot of using the experimental value of 4-5 x 10-6rn for g and the arbitrary values of 4 x lO4rn-1 and 6 for K and kl'lkl, respectively. The straight, dotted line cor- responds to the bimolecular, diffusional mechanism, with a value of 2 x 10s m-1 sec-1 for the constant. Obviously, the data are not compatible with the latter mechanism. The agreement between the curve and the experimental points is satisfactory ; indicating that the postulated equilibrium is a possible explanation of the experimental results. Additional measurements are required to test the hypothesis. In particular- the effects of temperature and of the viscosity of the solvent should be studied.Should further experimentation establish that this is the mechanism of the quenching action of retinene, it will be of real interest to determine what other quenchers act in this way and if any of chlorophyll addition compounds have their red absorption bands shifted to longer wavelengths. Steps of this type have frequently been proposed as essential links in the mechanism of photosynthesis, but they still lack direct confirmation. 1 + Wl'/kl - 1 ) m [ 1 + K(r + &!)I}, This research was supported jointly by a grant from the Graduate School of the University of Minnesota and by the United States Air Force through the Air Force Office of Scientific Research of the Air Research and Development Command under Contract No. 18(600)-1485. 1 Livingston and Owens, J. Amer. Chem. Soc., 1956, 78, 3301. 2 Livingston, J. Arner. Chem. SOC., 1955,77,2179. 3 Fujimori and Livingston, Nature, 1957, 180, 1036. 4 Rosenberg, Takashima and Lumry, Research in Photosynthesis (Interscience, New 5 Latimer, Bannister and Rabinowitch, Science, 1956, 124, 3222. 6 Livingston, Watson and McArdle, J. Amer. Chem. SOC., 1949, 71, 1542. 7 Weber and Teale, Trans. Furaduy Soc., 1957,53, 646. 8 (a) Watson and Livingston, J. Chem. Physics, 1950, 18, 802. (b) Weil, Diss. (Uni- 9 Brody and Rabinowitch, Science, 1957, 125, 555. 10 Forster, Fluoreszenz Orgunischer Verbindungen (Vandenhoeck and Ruprecht, 195 l), 11 Arnold and Meek, Arch. Biochem, Biophys., 1956, 60, 82. 12 Linschitz and Sarkanen, J. Amer. Chem. SOC., 1958, 80,4826. 13 Terenin and Ermolaev, Trans. Furaduy Soc., 1956, 52, 1042. 14 Fujimori quoted by Livingston in Handbuch der Pflunzen-physiologie, vol. V (Springer 15 (a) Brugger and Franck, Arch. Biochem. Biophys., 1958,75,465; (b) Franck, Proc. 16 Hubbard and Wald, J. Gen. Physiol., 1952, 36, 269. York, 1957), pp. 85-88. versity of Minnesota, 1952). pp. 243-255. Heidelberg, 1960). Nat. Acud. Sci., 1958, 44, 941.

ISSN:0366-9033    

DOI:10.1039/DF9592700144

出版商:RSC    

年代:1959

数据来源: RSC

摘要:

FLUORESCENCE YIELD AGAINST VELOCITY RELATIONSHIPS IN THE HILL REACTION OF CHLOROPLAST FRAGMENTS" BY RUFUS LUMRY, BERGER MAYNE AND J. D. SPIKES Dept. of Chemistry and Botany, University of Minnesota, Minneapolis, Minnesota, and the Dept. of Experimental Biology, University of Utah, Salt Lake City, Utah, U.S.A. Received 2nd February, 1959 The relative fluorescence yield of chloroplast fragments has been determined as a function of light intensity, temperature and concentration of Hill-reaction oxidant. Parallel Hill-reaction velocity measurements demonstrated a simple, direct relationship between the two phenomena. It is shown that the most elementary concept of a photo- synthetic unit, consisting of chlorophyll molecules for transfer of quanta and a quanta- trapping complex, is sufficient to explain intensity and temperature dependencies.The data are interpreted in terms of the efficiency of energy utilization, the manner of energy migration and the state of chlorophyll in vivo. It is concluded that fluorescence yields and lifetimes, as well as steady-state, Hill-reaction velocities, give direct information about the trapping complex. There are two major energy-transfer problems in photosynthesis. The first concerns the collection of light quanta from the photosynthetic pigments at those points where they are converted into some form of chemical or electric free energy. The second relates to the means whereby the units of useful energy thus trapped are condensed to produce oxygen. The collection of quanta has been extensively studied (see Rabinowitch 1 and Duysens 2).Such studies lead us with little doubt to the conclusion that resonance migration (sensitized fluorescence) plays a major if not the major role in the process. We then may think of the collection apparatus as consisting of a bed of pigment molecules whose major function is the reception and transmission of electronic energy, clustered about trapping centres for its conversion. It is but a short step to the concept of a photosynthetic unit consisting of the aggregate of chlorophyll molecules associated on the average with a single trapping centre and that centre. Of the various investigations supporting such a unit, perhaps the most conclusive have been the studies of the inhibiting effect of the 3-(chloropheny1)-1 : 1-dimethylureas by Wessels 3 and Spikes.4 For example, the dichloro compound (DCMU) completely eliminates the Hill reaction in concentrations of the order of one molecule of inhibitor for each several hundred chlorophyll molecules.We are thus in a position to ask searching questions about photosynthetic units. We should like to know the construction of the trapping centre and the details of its action; where and how the unused quanta are converted to heat; does the complete cycle of quanta trapping involve one, two or more quanta ; are all the chlorophyll molecules equivalent ; what is the efficiency of collecting system, and so on. The present communication provides partial answers to some of these questions and outlines methods which should provide information relating to all such questions. * This is paper number 4 in the series " The Mechanism of the Photochemical Activity of Isolated ChloropIasts".149150 HILL REACTION OF CHLOROPLAST FRAGMENTS In earlier work from our laboratories, it has been shown that the experimental steady-state rate law of the Hill reaction for light intensity is invariably of the form - kDIm V(ve1ocity) = - (m = concentration), I + kLlkD in which the average intensity 7 occurs because the chloroplast fragments average the intensity through successive excitation processes as they move through the rapidly stirred reaction mixture. Lumry, Rieske and Spikes 5 have shown that (1) may be interpreted to give the microscopic rate law applying at a single photo- synthetic unit in which I is the local intensity.Their further analysis led to the following re- action mechanism for the Hill reaction : {Tm) ++ 0 2 + T (4 a is the absorbance per chlorophyll molecule and n is the average number of chlorophyll molecules per photosynthetic unit. kt is the average trapping con- stant. T, the trapping centre, provides the point at which light quanta leave the singlet excited state of chlorophyll. The {Ti) are various intermediate forms of the cyclic transformations, probably chemical, of the trapping complex. There may be one such or many. Reactions (3d) are so fast as to be unobservable in steady-state velocity studies so that the velocity is dominated at both high and low light intensity by reactions of T in some form. The mechanism is incomplete in that " hidden " plant reactants, heat production steps and cross connecting steps between photosynthetic units are not shown.Since the dominant elementary step of the forms {Ti} is not known, (3) may be condensed to and kL = klm. If (3') is correct, T must be expected to act as a quencher for fluorescence from the singlet excited state of chlorophyll a, a situation which can be tested by measurements of fluorescence lifetime or fluorescence yield as a function of light intensity, for according to (3'), [TI = 1 - ~ / ~ n l a x . (4) Thus if the processes involving excited chlorophyll molecules, C, are C* 2 C + heat (c) Cf + T 2 (TI + C (d), ( 5 ) we may directly express the fluorescence intensity per photosynthetic units, dIf/dU, as a function of velocityR . LUMRY, B. MAYNE AND J .D. SPIKES 151 but in so doing it must be noted that ( 5 4 is not a mass-law matter. The lifetime of a quantum in the chlorophyll excited singlet state is of the order of 10-9 sec. The so-called working time of T, which is l/kD, is closer to 10-2 sec, so that there is no more than one photon undergoing migration in a photosynthetic unit at any one time. kt measures the average probability that it will get to T, and is, of course, relative to kf and kh. Then according to (3'4, ktunI is just the rate at which quanta can arrive at T if it is open and thus is the effective rate constant for (3'a). In the event T is open, the probability that an entering quantum will reach T is kt/(kh + k f + kr). Alternatively, when T is closed (in form (T}) the quantum can only be remitted as light or lost to heat with probabilities kf/(kh + k f ) and k d ( h + b).Eqn. (6) may now be converted to a function of the local light intensity using (2), the fluorescence yield 4 must then be expected to have the form, ( A is a constant) which predicts that 4 = (& - 4 J 2 , in which 40 is the yield at zero intensity, will occur at I = k,/k,. The latter quantity is also the light in- tensity for half-maximum Hill-reaction velocity. The proof that this is indeed the case for the Hill reaction is the principal aim of this paper. EXPERIMENTAL Tn order to obtain the same optical arrangement for fluorescence and Hill-reaction rate, both measurements were made in the same cell. Light from an incandescent source and passed by blue filters (Corning No.5433, centred at 440 mp) plus 1 cm of acid copper sulphate solution, fell on the fluorescence cell along the normal to its face. The emitted fluorescence from this face was intercepted by a photomultiplier (R.C.A. 621 7) placed at 45" to the normal. A complementary red filter (Corning No. 2412) was placed before the photomultiplier. Calibrated screens were used to vary the light intensity. The cell was mounted on a turret which could be turned to bring either a fluorescence standard which consisted of red gelatin mounted in glass or a vacuum thermocouple (General Electric radiation type 2A-4) into the light beam at the position of the cell. Fluorescence readings were always compared with those from the standard which was found by com- parisons with a primary standard of eosin-dye solution to be linear in the light dependence of its fluorescence intensity over the full intensity range of these experiments.Relative fluorescence yields are reported as the ratio of the photomultiplier-recorder deflection of the standard to that of the chloroplast suspension. The reaction velocity was measured by the potentiometric method of Spikes 6 et aZ. in the fluorescence cell which was formed of Lucite sheets so as to contain a cylinder of suspension 20mm in diameter and 2-1 nim in height along the light axis. Calomel and platinum electrodes were introduced through holes drilled in the cell top and stirring was achieved with the use of a vibrator-driven, stainless-steel wire which entered through another such hole. The cell was mounted on a large plastic chamber through which thermostatting water was pumped so that a temperature control of about 0.2 deg.was maintained. Because of experimental difficulties most of the fluorescence measurements were made on unstirred samples. In velocity measurements the stirring was at a high rate such that appreciable changes in rate did not alter the measured velocity. There was a transient phase in fluorescence of several seconds' duration at the beginning of illumination during which the fluorescence intensity moved asymptotically upward toward a constant value. The direction of this transient is consistent with this discussion. The chloroplast fragments were prepared by the method of Bishop et aZ.7 which pro- vides material nearly free of intact chloroplasts.The fragments were dispersed in water from which samples sufficient for one velocity or light measurement were withdrawn and frozen. Just prior to an experiment one such sample was thawed by a standard152 HILL REACTION OF CHLOROPLAST FRAGMENTS procedure, mixed rapidly with the other components of a reaction mixture and placed in the fluorescence cell. Readings were then made after a 5-min period for temperature equilibration. In addition to the oxidant, reaction mixtures contained 0.17 M sucrose, 0.01 M potassium chloride and phosphate buffer at pH 6.85 (0.05 M in total phosphate ion). RESULTS AND DISCUSSION THE FLUORESCENCE YIELD-VELOCITY RELATIONSHIP Typical results of a fluorescence yield study are given in fig. 1. Fig. 2 contains the companion velocity against intensity data.Here, as elsewhere, the precision in velocity determination is high, considerably higher than that of fluorescence, so that the rate law parameters can be determined with good reliability. In order to compare fluorescence with velocity, (6) must be modified so as to apply to the experimental arrangement. 1 2 4 I 0 P / O I' I I I u 0 I 2 3 log I0 FIG. 1 .-Relative fluorescence yield of Poke-weed chloroplast fragments (Phytoluccu americana L.) as a function of light intensities. Experimental conditions: 9"; 2 x 10-4 mole &Fe(CN)6 ; 43 mg/l. chlorophyll. FLUORESCENCE FROM A STIRRED SUSPENSION.-AS shown previously, the Hill reaction " sees " an average light intensity, Jo in which a is the cell depth, I0 the incident intensity and no the number of chloro- phyll molecules per cm3.Although terms such as exp (- ctnoa) were not negligible in these experiments (anoa = 3.8 for fig. 1 and 2), the analysis remains unchanged. We shall thus ignore them in this treatment.* r i s the effective local intensity for * If the light intensity at the rear wall of the cell is not a negligible fraction of 10, i.e. if terms such as exp (- anou) are not much smaller than unity, (11) becomes in which 81 = 1 - exp (- anoa); S2 = 1 - exp (- flnoa); 83 = 1 - exp [-@+a)noa]. Then I O ( ~ V ) = lo($+) = kDnoU/k&.R. LUMRY, B . MAYNE AND J . D. SPIKES 153 velocities at all x. Setting kj/(kh + k ~ ) = y, (7) becomes and is the intensity of fluorescence from a photosynthetic unit at point x along the direction of light propagation. If there are dU/dx = pdx photosynthetic units in a slab of unit cross-section and depth dx, the total fluorescence from this 10 kD = 0.119; kL = 0.048; K = 25.FIG. 2.Uill-reaction rate ; reaction conditions were the same as for fig. 1. dJfdU slab is -.-. As this radiation passes through the suspension toward the de- dU dx tector, it will be attenuated by absorption and scattering, the factor for which is /I per molecule. The total fluorescence emerging from the front of the cell is then and (1 1) The latter is a relative value with all geometric parameters and y thrown into a constant S. From (1 1) we find + a B ktnIo + k,noa154 HILL REACTION OF CHLOROPLAST FRAGMENTS Since on integration through the cell depth, the measured velocity is the half-maximurn velocity occurs at which is identicat with (14).IO(4V) = k,noQlktn, FLUORESCENCE FROM A RIGID SUSPENSION.-Eqn. (7) becomes (cf. (7'). Eqn. (7") may be multiplied by p and integrated to give general series solutions for I0 large or I0 small. /3 measures the average sum of attenuation of the light beam by turbidity and absorption at the fluorescence wavelengths. ci measures only the absorption of the incident beam since, to a first approximation, scattering will not attenuate the local field. Although neither of these quantities could be determined exactly, experiments with the opal-glass technique of Shibata et aZ.20 demonstrated that a and /3 were approximately equal in our solutions. Eqn. (7") may be integrated for this case of equivalence to yield the closed-form expression (17) : q3 = S k [ l - exp (- 2anoa)l - ktB[l - exp (- an~u)] + ktB2 In [ B + exp(--otnou) .In this case, kD in which B = - ktunlo S = $1 - exp(- 2anOu)l; and kD ktan lo(+$) = 0.9 - for values of exp (- anoa) of 0.20 and smaller. The expected test ratio (see below) for this case is, then, In comparing velocity and fluorescence yield, it is convenient to plot the quantities (4 - +o)/(&, - 40) and V/V,,,. 40 can usually be obtained ciirectly from plots of 4 against log Io. In many experiments, I0 cannot be made suffi- ciently high to provide directly good estimates of &,. As an alternative, we have plotted 1 I($ - $0) against 1/10 in these experiments, always obtaining good straight lines which may be extrapolated to 1/10 = 0 with small error.The linearity of this type of plot established the adequacy of (1 1) at least in form. For convenience, it is desirable to use (21) as has been done in fig. 3, a replot of data from fig. 1 : (21) 4 - do I0 dm - do - I0 + Io(+$)' -- Since 40 and are empirical quantities employed in fitting (21), the tests of the predicted mechanism and the equations for total fluorescence yield derived from it are reduced to tests of equational form and a single quantitative comparisonR. LUMRY, B. MAYNE AND J . D. SPIKES 155 of velocity and yield curves. All yield and velocity data obtained in these experi- ments were well fitted by the predicted expressions. As the remaining test we have used the quantity Zo($+)/Zo(+V), which we call the test ratio.A few successful measurements of full yield-intensity curve using stirred sus- pensions gave data following (21) and thus (11). The test ratio was unity within error at higher fragment concentrations and decreased slightly with concentration. Eqn. (17) cannot be distinguished from (21) within present errors. Data secured at 9" from an unstirred suspension follow (21) with good accuracy, as shown by the solid line of fig. 3. The velocity data expressed as V/Vmx lie on a solid line of exactly the same form but lying slightly to the left, so that the test ratio is 1-52. I I I 1 I I I I log I0 2 x 10-4 M K3Fe(CN)6 40 dal 0 9" 0.77 3-35 0 20" 0.78 3.50 FIG. 3.-Relative fluorescence yield of Poke weed as a function of light intensity and temperature, plotted according to (17) ; the 9" data from fig.1. As demonstrated in fig. 4 and 5, at higher suspension concentrations (larger no) the test ratios were unity. According to (20), the ratio should drop from 0.45 in fig. 3 (43 mg/l. chlorophyll) to 0.14 in fig. 5 (143 mg/l. chlorophyll). Quite clearly our model of a motionless system is not appropriate for integration of the fluor- escence in an unstirred system in which Brownian motion may cause considerable light averaging. Thus, although yield-intensity plots have the correct form and the measurements in stirred systems, where the intensity-averaging process is better understood, are consistent with our initial hypothesis about light trapping, it was necessary to turn to temperature studies for further proof. EFFECT OF TEMPERATURE Since the only temperature-dependent parameter in our analyses is kD, yield and velocity should show the same temperature dependence. The 9" experiments were repeated at 20" (results plotted in fig.3) to provide a test ratio of 1.64, which is identical with the 9" value within small experimental error. This fact, combined with those previously mentioned, form strong preliminary support, though certainly156 1.0 9 h 0.8 I 0.6- 8 2 I z 1 n 3 0 - 4 0 I I I I I I - - - /: / - - OS2;/; 2 , , 3 , 1 I 0 I 2 3 log (10/72.5 ergs cm-2 sec-1) K3Fe(CN)6 prep. conc. do #a A 2 3 x 1 0 - 4 ~ 0.84 4 0 a 1 1 x 10-3 M 1.0 4.2 0 1 0 1.0 4-2 A 2 0 0.84 4.0 FJG. 5.-Relative fluorescence yield of Poke-weed chloroplast fragments as a function of light intensity and oxidant concentration.The solid line at 3 x 10-4 M &Fe(CN)6 was calculated from velocity data. Experimental conditions : 143 mg/l. chlorophyll ; 15".R . LUMRY, B. MAYNE AND J . D . SPIKES 157 not absolute proof, for the proposed trapping system. On this basis, we may now turn to a consideration of the consequences of the model. SOME IMPLICATIONS OF THE MODEL The adequacy of (1 1) implies that both fluorescence and velocity measurements of the steady-state Hill reaction measure directly reactions of the trapping complex and that the light dependence is such that we may assume the period of the trans- formations of the trapping complex to be long with respect to the lifetime of a quantum in the chlorophyll bed. If the latter situation did not obtain, quanta untrapped in their first passage to the trapping centre would be able to return for a second chance, and the rate of trapping, and thus the velocity, would not be a rectangular hyperbolic function of light intensity.Strictly speaking, of course, some quanta may be trapped on second or third passage, but the fraction which is, is much too small to be detected (see Introduction). Since it is unlikely that quanta in excited chlorophyll molecules move from the first excited singlet state to some other electronic state and then return to the excited singlet, the quanta must remain in the latter state at all times before trapping. If not trapped, they are lost to fluorescence and heat and since there must be a heat loss process from the singlet,g (11) insures that the major loss reaction is of the form of (5c).Apparently the latter step involves a different internal conversion process than that normally observed in organic solutions, since Rosenberg and co-workers 8 could not detect the chlorophyll triplet state in chloroplast fragments and Livingston9 has put the lifetime of the excited singlet at less than Brody’s value19 of 1.6 x lO-gsec, which is about four-fold shorter than observed in organic solutions under conditions of no self-quenching. It may be that the immediate local environment of the chlorophyll molecules modifies the excited states to improve internal conversion for heat production. On the other hand, there may be occasional pairs of chlorophyll molecules which provide a self- quenching site of the type proposed by Forster.17 However, it is clear that the only fast quenching process is trapping itself.Thus the organization of the plant guarantees the virtual absence of ordinary self-quenching. Most quanta not used for oxygen production are lost in process (54, as is shown by the large relative size of &, to 40 (fig. 3). Loss processes after the quanta reach T are probably minor. The appearance of $0 different from zero may be attributed to one or both of two causes. Trapping is maximally efficient at zero light intensity but this efficiency is not one-hundred per cent, either because as a consequence of normal morphology or damage during preparation some chlorophyll are different from others in the sense that they have no trapping centre; or, on the other hand, be- cause geometric considerations are such that the average photon has a non-zero chance of loss during migration before it reaches the trapping centre.Available information does not allow a distinction in this matter but it can be decided by further studies of preparative, ageing and inhibitor variables. Eqn. (11) is compatible with a trapping centre consisting of an ordinary chlorophyll molecule and a plant reactant E such that resonance migration to T = Ct E is quantitatively the same as migration to any other chlorophyll molecule in the bed. Since in our experiments on Poke-weed the lifetime in the excited state may increase by as much as a factor of 4 at high Hill-reaction velocity, an average quantum might then migrate to B] several times, passing back to the bed each time.This situation requires the trapping sequence Ct + c* -+ Ct* + c (a) migration, c + C** + c* + Ct (b) back migration, C t + E + T (c) pre-formation of T, C* + T -+ { E ) + Ct + C (22) ( d ) trapping.158 HILL REACTION OF CHLOROPLAST FRAGMENTS Alternatively, (11) does not exclude the possibility that T, whether it is composed of E plus Ct or quite a different pigment molecule, has a spectral region lying to the red side of the chlorophyll red peak, thus improving its efficiency in the com- petition for quanta. It has not yet been possible to apply our method to test the latter possibility due to Franck.10 HEAT LOSS CORRECTION TO THE QUANTUM REQUIREMENT The actual energy requirement of the Hill reaction is undoubtedly nearly identical with that of photosynthesis.18 The minimum quantum requirement per oxygen molecule of the Hill reaction is no greater than 8.18 In determining the latter value, no correction was made for quanta lost to heat and fluorescence.log (Zo/72.5 ergs cm-2 sec-1) K3Fe(CN)6 4 0 4m conc. 0 0 0.60 1.75 0 1 0 - 4 ~ 0.60 1.71 10-3 M 0.67 1.74 10-2 M 0.45 1.05 FIG. 6-Relative fluorescence yield of sugar-beet chloroplast fragments as a function of light intensity and oxidant concentration. Experimental conditions : 82 mg/l. chlorophyll ; 15". If fluorescence yields are measured in a companion experiment, the corrected quantum requirement, which we may call the " trapped " quantum requirement Rt, since it alone correctly measures the efficiency of utilization of trapped energy, is obtained by This correction may not be negligible. In the five fragment preparations used in these experiments, the factor had the values sugar beet : 0.55 and 0.53 ; poke weed : 0.76, 0.78 and 0.76.R. LUMRY, B.MAYNE A N D J . D. SPIKES 159 Although it would thus appear that each type of plant has its own constant factor, the preparative procedures were so nearly identical that the observed constancy might be expected on that basis alone. Nor can it be assumed that the minimum requirement for sugar beet is 8 x 0.55 = 4 since the correction factor was deter- mined in the present work at a pH far removed from the pH of minimum require- mentls (6.3) and using blue light, which is some 35 % less efficient than red light.5 THE EFFECT OF OXIDANT Fig. 5 and 6 demonstrate the dependence of fluorescence yield on ferricyanide concentration.Previous studies 11 of reaction velocity demonstrated an increase in k, and a decrease in k, with increasing oxidant concentrations. The figures support these observations and allow a deeper analysis of the k, effect. At 10-3 M ferricyanide and below, $0 and &, are independent of oxidant. At 10-2 M, $0 and 4m are considerably decreased to provide for sugar beet a quantum-requirement correction of 0.1. This large correction indicates that most of the reduction in k, is due to direct quenching by the oxidant, rather than a decrease in kt. Either effect would produce the observed decrease in the rate parameter kL, but only direct quenching according to (1 1”) explains the fluorescence observations.Here (11”) Kq is the quenching constant of the oxidant. The effect of ferricyanide on k, probably reflects the decoupling of photophosphorylation and the Hill reaction by the oxidant.12 Since the only oxidant present in appreciable concentration in the absence of ferricyanide is probably oxygen, it is tempting to interpret the leftmost curve of fig. 5 and 6 as measuring the behaviour of oxygen as a Hill-reaction oxidant Further study will be necessary to establish this point. CONCLUSION There are undoubtedly other ways in which to explain the fluorescence yield data reported here. For example, Franck‘s 1 narcotic mechanism could probably be made to work. In view of the simplicity and adequacy of the present analysis, recourse to such alternatives does not appear to be necessary (see, for example, (13) and (14)). The compatibility of some of our conclusions with those based on data from whole photosynthesis of intact cells can be judged by reading R.S . Livingston’s contribution to this symposium. In general, we conclude that most chlorophyll molecules in grana behave much as they do in organic solution, with the exception that self-quenching is eliminated. The possibility that T contains a chlorophyll molecule thus given special properties is considerable, as judged by reports such as that of Vishniac and Rose.15 It now seems possible that the so-called “ 254 mp ” coenzyme Q 16 is also a constituent of T. It may, in fact, develop that the trapping process requires some complex of small though exotic organic molecules, protein and lipoid serving only for geometrical arrangement. Whether or not enzymes are necessary participants in the reactions of (T} is not yet clear.The small tem- perature-dependence of k, and the thermal inactivation studies of Bishop et aZ.7 certainly suggest this fact. However, rearrangements of atoms or electrons in a nonproteinoid trapping centre could also demonstrate a small temperature de- pendence. Crystal-like semi-conduction is, however, not indicated in the col- lection of quanta at T, nor does it appear to be compatible with the pattern of data pertaining to k, which has accumulated. It now appears fairly certain that an encyclopedia of information about the reactions of a single functional element of the entire photosynthetic process, the1 60 HILL REACTION OF CHLOROPLAST FRAGMENTS trapping complex, can be constructed from measurements of fluorescence yield, fluorescence lifetime, and the Hill-reaction velocity of chloroplast fragments. This fortunate situation should prove useful in future studies of photosynthesis. We are indebted to the United States Atomic Energy Commission, the National Science Foundation, and the Office of Naval Research (NR-104-030) for the support which made this work possible. 1 Rabinowitch, Photosynthesis and Related Processes (Interscience Publishers Inc., 2 Duysens, Diss. (University of Utrecht, Netherlands, 1952). 3 Wessels and van der Veen, Biochim. Biophys. .4cfa, 1956, 19, 548. 4 Spikes, Plant Physiol., 1956, 31, xxxii. 5 Lumry, Rieske and Spikes, in press, Plant Physiol., 1959. 6 Spikes, Rieske and Marcus, Plant Physiol., 1954, 29, 161. 7 Bishop, Lumry and Spikes, Arch. Biochenr. Biophys., 1955, 58, 1. 8 Rosenberg, Takashima and Lumry, in Research in Photosynthesis (Interscience 9 Livingston, this Discussion. 10 Franck, Daedaliw, 1955, 89, 17. 11 Lumry and Spikes, in Research in Photosynthesis (Interscience Publishers Inc., 12 Avron, Krogmann and Jagendorf, Biochim. Biophys. Acta, 1958,30, 144. 13 Shiau and Franck, Arch. Biochem., 1947, 14,253. 14 Kautsky and Zedlitz, Naturwiss., 1941,29, 101. 15 Vishniac and Rose, Nature, 1958,182, 1089. 16 Bishop, personal communication. 17 Forster, Fluoreszenz Organische Verbindungen (Varidenhoeck and Ruprecht, 195 1). 18 Lumry, Wayrynen and Spikes, Arch. Biochem. Biophys., 1957, 67,453. 19 Brody and Rabinowitch, Science, 1957, 125, 555. 20 Shibata, J. Biochem. (Japan), 1958, 45, 599. New York, N.Y., 1951), vol. 11, part 1. Publishers Inc., N.Y. 1957), p. 85. N.Y., 1957), p. 373.

ISSN:0366-9033    

DOI:10.1039/DF9592700149

出版商:RSC    

年代:1959

数据来源: RSC

摘要:

PRIMARY PHOTOCHEMICAL AND PHOTOPHYSICAL PROCESSES IN PHOTOSYNTHESIS BY EUGENE RABINOWITCH Dept. of Botany University of Illinois Urbana Illinois Received 9th March 1959 The structure of chloroplasts and the optical properties of chlorophyll are examined from the point of view of possible migration of excitons and separation of charges after light absorption in a chlorophyll molecule. Evidence suggests the presence in chloro-plasts of non-crystalline monomolecular chlorophyll layers. It also appears that the chlorophyll molecules are present in (at least) two different states. Resonance migration of excitation energy over 100-200 chlorophyll molecules appears plausible ; perhaps these are molecules attached to a single globular protein molecule in the chloroplast lamellae.There is no spectroscopic evidence for the presence of electron conductance levels ; argu-ments for their existence derived from paramagnetic resonance experiments do not seem convincing. Evidence suggesting that chlorophyll molecules undergo reversible trans-formations (perhaps involving a reduction) in photosynthesis is discussed ; the two types of chlorophyll present in chloroplasts may be involved in two different steps of the primary photochemical process. EXCITONS AND THEIR MIGRATION In photochemical reaction in gases we distinguish between the primary photochemical process and the secondary reactions which in themselves do not require light. For example in the formation of HC1 from C12 + H2 the primary photochemical process is the dissociation of C12 into Cl atoms and the secondary reactions are C1 + H2 -+ HC1+ H and H + C12 + HCl + C1.In photochemical reactions in condensed molecular systems (we leave aside extremes of ionic or metallic crystal lattices) additional step (or steps) can occur between the light absorption by a molecule and the primary photochemical re-action the “ photophysical ” processes of energy or charge transfer and energy or charge migration (by repeated transfer). In one extreme case the whole system acts in the excited state as a single giant molecule; either the excited electron, or the hole it leaves in the electronic cloud of the absorbing molecule or both, are in this case not localized but belong to the system as a whole. In the less extreme case the excited electron and the hole are associated at any given time, with a definite molecule the period of this association being long compared to intramolecular vibration periods ; however during the electronic excitation period, the electron or the hole or both can be transferred to another nearby molecule.Under appropriate conditions this transfer can be repeated many times before the electronic excitation is dissipated so that the electron or the hole or both, may be found at the end of the excitation period in a site far removed (on molecular scale) from the original locus of excitation. If the electron and the hole travel separately (or if only one of them travels) the final effect is separation of charges-which can be also described as internal oxidation-reduction. If they travel together the final effect is the migration of excitation energy without separation of charges.The electron-hole pair is often referred to as an exciton.1 When the excited electron and the hole are associated in a single molecule the (perhaps superfluous) term “ intramolecular exciton” is used in solid-state physics. In this case the transfer of the exciton to a neighbouring position in the “ lattice ” formed by F 16 162 PRIMARY PROCESSES I N PHOTOSYNTHESIS the condensed medium can be achieved in two different ways-either by the simultaneous transfer of two electrons in opposite directions (b in fig. 1); or by the return of the electron in the " donor " molecule in cell I to its normal state, and simultaneous excitation of the electron in the " receptor " molecule in cell I1 ( a in fig.1). Actually mechanism (a) and (6) both contribute to the transfer in either " coincident " or " distant " electron-hole pairs (" intramolecular " or " intermolecular " excitons) but with different relative probability ; mechanism (b) being most important in the first case and decreasing in importance as the distance between hole and electron increases (fig. l(c)). F] 0 1.7.1.1 FIG. 1 .-Transfer of intramolecular and intermolecular excitons. 0 hole; electron; * excited electron (A) Transfer of intramolecular exciton-Forster-Heller-Marcus mechanism. (B) Transfer of intramolecular exciton-Wannier mechanism. (C) Conversion of intra- into intermolecular exciton its transfer and dissociation. The binding energy of the exciton may be so small that it will easily dissociate, either under the influence of thermal motion or under the pull of an applied electric field.In both cases the system will act as a " photoconductor ',. If electron acceptors or electron donors are present as impurities in the system or adsorbed on its surface their affinity for the electron or the hole may trap the one, or the other or both. The mechanism of photobiological processes such as photosynthesis in plant cells has been customarily approached from the side of the experience gained in the study of photochemical reactions in gases or dilute solutions. Recently how-ever it has been pointed out that the concentration of pigment molecules in the photosynthetic organelles of plants (or visual organelles in animals) is so dense as to justify approaching photosynthesis (or vision) from the point of view of the phenomena in the solid state.To discuss this approach we must first summarize the present knowledge of the actual structure of the photosynthetic apparatus. 2. STRUCIWRE OF CHLOROPLASTS With the exception of the primitive blue-green algae (Cyanophyceae) and of purple and green bacteria all photosynthesizing plants contain chloroplasts-subcellular organelles which contain the green pigment chlorophyll as well as various other " accessory " pigments (carotenoids phycobilins). These organelles are the sites of photosynthesis and their internal structure appears to be significant for the mechanism of this energy-storing process FIG. 2.-Granular chloroplasts in mesophyll cells and lamellar chloroplasts in maize (Zea mais) (after Vatter).[To face page 162 FIG. 3~.-Intergranular lamellae in a granular chloroplast of maize (Zea mais) (after Vatter) FIG. 3e.-Intergranular lamellae and intragranular (after Vatter) FIG. 6.-Macromolecules in intragranular lamella E . RABINOWITCH 163 All chloroplasts contain lamellae-flat or involuted thin extended layers, with a thickness of 100-2OOA. Such lamellae are found even in chloroplast-free cells of the blue-green algae ; they probably exist also in photosynthesizing bacteria. The lamellae are arranged in more or less orderly parallel systems. Two main types of chloroplasts are known in one the lamellae fill out more or less uniformly the whole body of the chloroplast (" lamellar " chloroplasts) ; in the other they form cylindrical grana (" granular " chloroplasts).Fig. 2 and 3 show both types in the came plant-maize; the granular chloroplasts are found in the cells of the lea' esophyll the laminar chloroplasts in the vascular bundle cells. In a general wa! t can be said that granular chloroplasts are common in mature cells of the hig; land plants while lamellar chloroplasts are more characteristic of younger cell nd of the algae. i . 2 shows that in granular chloroplasts too the lamellar structure extends thro !bout the whole chloroplast including the intergranular " stroma " ; the grxLL are merely approximately cylindrical volumes in which the lamellae are denxl- and more numerous (probably in consequence of splitting of a single inter-granular lamella into two or more lamellae inside the granum).The chloroplasts differ from the protoplasm in their lower content in proteins (- 60 % instead of > 95 %) and higher content (- 40 %) of " lipoids " i.e. compounds soluble in ether and alcohol. The latter includes up to 5 % pigments -chlorophyll and the carotenoids ; the red and blue " phycobilins " are however, attached to proteins and can be extracted with water. The high optical density of the grana and the lamellae in electron micrographs of preparations fixed with osmic acid (as in fig. 2 and 3) is due to higher concen-tration of precipitated osmium oxide; and this in turn seems to be due to the presence of lipoids (although this relation is a matter of some controversy between electron microscopists).In other words the dense lamellae seem to contain the greatest concentration of the lipoid constituents while the interlamellar layers of the stroma are more predominantly proteinaceous (more cautiously one can say that the lipoids in the lipoproteids of the lamellae contain more active groups than those in the interlamellar stroma). For granular chloroplasts absorption and fluorescence microscopy confirm that the pigments (in particular the chloro-phyll) are located in the grana. [Hodge suggested (cf. fig. 4a) that the intergranular lamellae also carry the pigment i.e. that the grana are a darker green and the stroma a fainter green but not colourless; it seems however more likely that when lamellae become organized in grana the pigments are coiicentrated more or less completely in the latter as in fig.4b.1 It has been suggested that chlorophyll whose molecule consists of a flat coloured conjugated ring system (chlorin) and a long colourless almost saturated tail (phytol), should have the tendency of forming monolayers on interfaces between hydro-philic (proteinaceous) and hydrophobic (lipoid) layers. Preliminary estimates 293 indicated that the total surface area of the lamellae in a typical chloroplast is about adequate for uniform monomolecular distribution of the chlorophyll available in the chloroplasts allowing 100-200 A2 per molecule-which is approximately the area required by the flat chromophore " head " of the chlorophyll molecule. Thomas and co-workers 3 carried out somewhat more precise comparisons of the amount of chlorophyll present in the chloroplasts of different species and the surface areas of their lamellae (counting only those within the grana).The results were as follows Spinacia cleracea 3.2 mp2 per molecule ; Hibiscus rosa sinensis, 3.6 mp2 ; Aspidistra elatior 0.9 mp2 ; TuIipa spec. 1-8 mp2 ; Elodea densa 2.5 mp2 ; Mougeotia sp. (grana-free) 3.8 mp2 ; Spirogyra sp. (grana-free) 2.5 mp2 ; Syne-chococcus cedrorum (chloroplast-free cells) 0.8 mp2 ; Nitzschia dissipata (grana-free) 2-5 mp2. At the same time the amount of chlorophyll per chloroplast varied widely-between 7 x 10-14 g and 3.4 x 10-9 g. Artificial chlorophyll monolayers 53 6 exist in two forms crystalline and amorphous. In the first form the area requirement is of the order of 0.75 mp 164 PRIMARY PROCESSES IN PHOTOSYNTHESIS (suggesting that the layer may be bimolecular rather than monomolecular) and the red absorption band peak is shifted from 660 mp in solution to 735 mp in the monolayer (fig.5) ; in the second form the space requirement is about 1-06 mp2, and the red absorption band is located at about 678 mp. This shows that chloro-phyll in the living cells (where its absorption maximum lies at 675-680mp) does not form crystalline monolayers (or three-dimensional crystals which have a band peak at about 740 mp) ; it could be conceivably present in the form of amorphous m onolayers. GRANUM 250 A V FIG. ~A.-Two interpretations of the granular structure of chloroplasts. C chlorophyll ; L lipoids ; P protein A. Granular chloroplast after Hodge et al.(J. Biochem. Cytol. 1955 1 605). 0 RANUM STROMA 2 5 0 2 7 0 FIG. 4~.-Granular chloroplast after Steinmann and Sgostrand (Expt. Cell. Res., 1955 8 15). lipoproteid ; lipoproteid carrying chlorophyll In both types of artificial monolayers the chlorophyll ring systems must be stacked obliquely since the actual area of this system-which would be required for the molecule to lie flat on the interface-is as high as 2.4 mp2. The electron micrograph of granular chloroplasts suggest the presence in the latter of flat " chambers " enclosed between pairs of lamellae (fig. 4a b). Swelling experiments on grana material support this picture (fig. 6) ; and it seems tempting to ascribe to these chambers an important function in photosynthesis-for example, one could suggest that one product of the primary photochemical oxidation-reduction reaction (e.g.the reduction intermediate) is trapped in the chamber, while the other (the oxygen precursor) escapes on the outer side of the chamber. However it must be kept in mind that lamellar chloroplasts containing no grana, and thus without similar " chambers " are perfectly capable of photosynthesis. Some evidence suggests that such continuous lamellae tend to form double layers ; the composition of the interstices between the lamellae could thus be different on the two sides and a separation mechanism of the above-suggested type could operate in the lamellar system as a whole with the interstices taking up alter-nately the primary oxidation products and the primary reduction products of the photocatalytic process E.RABINOWITCH 165 The use of the lamellar chloroplast structure to provide a separation of the oxidation and reduction intermediates in photosynthesis was first suggested by Calvin and co-workers 7 in conjunction with considerations of electron migration in such a structure making the chloroplast an analogue of Bell's " solar battery ". The dense arrangement of pigment molecules in the chloroplast lamellae does in fact invite speculations concerning possible energy and charge migration in these structures ; but the specific picture used by Calvin and co-workers does not seem to be the most likely one in consideration of what we know about the actual structure as well as of what we can surmise about the purpose of energy transfer in photosynthesis (see § 4).wavelength in mp FIG. 5.-Absorption spectra of chlorophyll monolayers colloidal (lower curve) and crystalline-probably a bimolecular layer (upper curve). Arrows show location of red band in solution (after Jacobs et d.). Swollen and collapsed intergranular " pockets ". Confirmation of the arrangement of chlorophyll in thin-probably mono-molecular-layers was provided by Goedheer's 8 studies of dichroism and bire-fringence of certain large chloroplasts. A relatively weak dichroism is observed in these chloroplasts together with remarkably strong selective birefringence (" birefringence dispersion "). In the interpretation of these results account must be taken of the undoubtedly present lamellar structure (" form dichroism " and " form birefringence ") and of the possibly existing pardel orientation of Zight-absorbing molecuZes (" intrinsic dichroism " and " intrinsic birefringence ").Goedheer concluded from various observations (including the effect of plasmo-lysis and of vital staining with rhodamin B) that the most likely interpretation of all observations can be based on the assumption of the presence in chloroplasts of very thin dense pigment layers (about 0.2-0.6mp thick) whose index of re-fraction is much higher than that of the protein or lipoid layers. In these-obviously monomolecular-chromophoric layers the axes of the chlorophyll molecules seem to be only very imperfectly oriented thus accounting for the relatively slight dichroism. Goedheer suggested that the imperfect alignment of molecular axes may be due to the fact that the monolayer covers not a flat but a " grained " surface, consisting of spherical macromolecules of lipoproteins visible on high-magniiica-tion electron micrographs of the lamellae (fig.7). (This of course is not the onl 166 PRIMARY PROCESSES I N PHOTOSYNTHESIS possible form of disorder in the monomolecular layer !) The macromolecules have a diameter of 7-1Omp. A sphere of this diameter has a total surface area of 150-300 mpknough to accommodate up to 150-300 adsorbed chlorophyll molecules ifit could be covered on all sides-which is difficult if the macromolecule is part of a lamella. n n ;:Tx 'Fm ---Chl. ' C hl 'Chl. A 8 FIG. 7.-Terms of fluorescent and non-fluorescent chlorophyll in solution. A.non-fluorescent state nn-level below rn-level ; B. fluorescent state nn-level above mr-level. In A practically all excited 1Chl* fall into triplet state via the nn-level ; in B about 30 % (in chlorophyll a) are re-emitted as fluorescence the rest presumably goes into the triplet state. 3. STATES OF CHLOROPHYLL IN THE CHLOROPLASTS Various evidence suggests that the chlorophyll molecules in the chloroplasts are present in two (if not more) different forms. Duysens 10 noted that the width of the chlorophyll absorption band in vivo in itself suggests the superposition of two bands. Krasnovsky and co-workers11 and Smith and Koski12 observed shifts in the position of the chlorophyll absorption peak during the greening process of etiolated leaves and parallel changes in the photochemical stability of the pigment and concluded that chlorophyll first arises in one form (Chl684), than changes into another (Chl 673) and possibly into a third form (Chl 677).It has been suggested that one of the forms present in green cells is monomeric and another one " aggregated ". Apparently discordant measurements of life-time of fluorescence in vivo by Brody 13 and of the quantum yield of chlorophyll fluorescence in vivo by Latimer 14 can be reconciled by the assumption that only about 3 of chlorophyll (in ChforeZfa) is in the fluorescent state (with a quantum yield of fluorescence approximately 10 %) while $ are non-fluorescent (giving an average fluorescence yield of 3 %). Other observations (cf. Brody 15) make it appear likely that one non-fluorescent form of chlorophyll in vivo (which however, can be present only in relatively small amounts because of the position of the main absorption peak at about 678 mp) has an absorption band at 705 mp and a corresponding fluorescence band at 720 mp ; the latter appears however only at low temperatures (in liquid air).It is to be noted that the " monomeric " and " polymeric " " fluorescent " and " non-fluorescent " forms of chlorophyll in vivo probably correspond to differ-ent states of identicalqmolecules in a monolayer-possibly distinguished by the density of the monolayer or by the kind of molecules with which they are associ-ated. Franck 16 concluded from the kinetics of photosynthesis that two types of chlorophyll in vivo are characterized respectively by their contact with water, or with exclusively non-polar " lipoid " molecules.The chlorophyll molecules in contact with water are fluorescent ; no free electrons can occur in this hydrated part of the monolayer. The lipoid-protected part of the monolayer is non-fluorescent and could support the existence of free electrons (demonstrated i E. RABINOWITCH 167 Arnold's experiments 17 on dried chlorophyll layers which may or may not be relevant to the conditions in live chloroplasts). In accordance with the analysis of the excited chlorophyll levels by Platt,l8 the order of the two types of excited states nr and mr may be reversed in the two forms with the non-fluorescent nn state being the lower one in " lipoid-bound " and the upper one in the hydrated (or aminated) form (thus accounting for their difference in fluorescence).Franck's suggestion is that in the primary photochemical process in photo-synthesis the co-operation of two excited molecules is required one of which must be in the nn excited state while the other c m be in the izr state. Therefore the primary process can occur only at water-exposed chlorophyll molecules ; however, the lipoid-protected molecules can contribute by energy transfer one quantum to the two-quantum primary process. 4. ENERGY AND CHARGE TRANSFER IN CHLOROPLASTS The problem of energy transfer arose in the study of photosynthesis in 1936, when Gaffron and WON 19 first attempted to interpret the results of Emerson and Arnold's fundamental experiments on photosynthesis in flashing light by the assumption that about 2000 chlorophyll molecules act in vivo as a " photo-synthetic unit "-a kind of energy catch-basin associated with a single " reduction site " in which the primary photochemical process takes place.Because of this association the maximum amount of oxygen that can be produced by a single flash of light corresponds to one molecule 0 2 per 2000 molecules of chlorophyll (this being the main finding of Emerson and Arnold). The number 2000 must be re-duced however-probably by a factor of 8-because the liberation of one molecule of oxygen requires not one but several-probably eight-identical elementary photochemical processes. The most likely number of chlorophyll molecules in the photosynthetic unit is therefore 2000/8 = 250. This brings the unit into the range where one could consider as possible its identification with the previously mentioned pigment-bearing macromolecules.The (hypothetical) picture of protein macromolecules carrying about 250 chlorophyll molecules is remarkably similar to the (empirically derived) picture of the phycobilin molecules extracted by water from red algae; the latter appear to be protein macromolecules with a molecular weight of about 280,000 carrying 50-100 chromophores ( S . and M. Brody 20). The difference between the phyco-bilins and the chlorophyll-complexes in vivo may consist mainly in the additional association of the chlorophylls with lipoids (probably by the lipoid affinity of the hydrophobic phytol " tail ") ; this association may be what prevents the chloro-phylls from being extracted by water in the form of stoichiometric complexes (as distinct from arbitrarily chopped fragments containing both the proteinaceous and the lipoidic constituents).When Gaffron and Wohl introduced the concept of the photosynthetic unit, they suggested that the co-operation between 2000 (or as we would now say 250) light-absorbing chlorophyll molecules and the single reaction centre (enzyme molecule) can be conceived in two different ways. One is to envisage " chemical messengers "-energy-rich particles generated photochemically at each light-activated chlorophyll molecule and migrating to the reaction centre. The other is to visualize energy migration by a chain of resonance transfers. It is the second picture that has fascinated workers in the field of photosynthesis in the last twenty years without a definite answer being found to the question of its relevance.For the phycobilin macromolecules Goedheer's measurements of fluorescence polarization (which proved to be very low) suggest that the excitation energy is re-emitted not by the molecule that has first absorbed it but by another molecule, with an orientation quite different from that of the first one. This and the experi-ence gained in the study of excitation energy migration in solutions and crystals (as well as theoretical calculations of the probability of the resonance transfe 168 PRIMARY PROCESSES I N PHOTOSYNTHESIS of energy) make it appear likely that the excitation energy moves around freely among the 50-100 chromophores attached to the protein macromolecules of phyco-bilin.In analogy one could expect an equally efficient exchange of excitation energy also between the chlorophyll molecules attached to a common protein macromolecule. It may be that the area of efficient energy migration takes in, not one but several adjacent macromolecules thus accounting for the relatively large number (at least 250) molecules in the “ kinetic” unit. However in this case one would have to assume that “ reaction centres ” are less numerous than the protein macromolecules while the natural assumption seems that the protein macromolecule itself (or a certain spot on it) is the enzymatic “ centre ”. The mobility of the excitation energy in the protein-chlorophyll (and protein-phycobilin) complexes may be incidental ; but-as mentioned above-it may also be a useful device for the effective utilization of the quanta absorbed in any one of the 250 chlorophyll molecules for the initiation of an enzymatic reaction chain.The plant cell cannot provide a separate enzymatic “ conveyor belt ” for each chlorophyll molecule simply because of lack of sufficient space. The high con-centration of chlorophyll in the cell (up to 10-2mole/l.) is needed to provide efficient absorption of light (no organic pigment could do significantly better). Even in direct sunlight each chlorophyll molecule absorbs quanta only about once every 0.1 sec. Enzymes on the other hand are known to handle their substrates in one-thousandth of this time or less. The cells thus face both the necessity and the possibility of reducing drastically the number of independent reaction paths when passing from the light-absorbing stage to the enzymatic stage of photosynthesis.Excitation energy migration in the “ photosynthetic unit ” provides an elegant answer to this problem. One could ask whether a special “ reduction ” mechanism is really necessary if the macromolecule carrying the 250 chlorophyll chromophores is itself the enzyme involved in the primary photochemical reaction. The question is related to the general problem why are the enzyme molecules so big although only a relatively small group in them is likely to be directly involved in the catalyzed reaction? Whatever the answer to this question it is likely that the electronic excitation energy must be brought to a specific spot on the macromolecule in order to be utilized for catalysis ; resonance migration mechanism is a plausible answer to this necessity.It has been asked (first by Franck and Teller 21) whether enough time is avail-able between light absorption in the chloroplast and energy dissipation (by fluor-escence or by one of the intervening quenching processes) to permit effective energy transfer to the reaction site in a 250-molecules unit. If the transfer is by “random walk” an average of considerably more than 250 transfers will be needed to assure effective “delivery ” of the quantum to the reaction centre. (On the other hand it may be sufficient to conduct the quantum to the general neighbourhood of the “sink ” rather than to the one single pigment molecule in direct contact with it.) The natural life-time of chlorophyll in the lChl*-excited state is 15 mp sec (calculated by integration of the absorption band cf.Brody and Rabinowitch 13). With Latimer’s 3 % fluorescence yield the actual life-time is 0-5 mp sec ; with Brody’s 10 % yield 1.5 mp sec. To make (say) lo00 energy transfers during this period means that a “ visiting time ” of < 0.5-1.5 x 10-3 mp sec is available for each visited molecule. The width of the absorption band of chlorophyll in vivo suggests undisturbed coupling with intramolecular vibrations, and this requires electronic excitation of the absorbing molecule to last > 10-4 mp sec. Comparison of the two figures (< 0.5-1.5 x 10-3 mp sec available and > lO-4mpsec needed for each visit) shows just about enough time for the postulated lo00 visits with practically no time to spare.This is a little too “ tight’’ for comfort. The range of effective migration could be considerably extended if one were permitted to assume (i) that the yield of fluorescence is limited not by complete dissipation of the quantum but b E. RABINOWITCH 169 transfer into the metastable state- 3Chlwhich is plausible and (G) that energy migration can continue after this transfer by the " Wigner mechanism " Khl 4-3Chl, + 1Ch1 + 3Chl (with conservation of total spin)-which is somewhat doubtful. Resonance energy transfer in the triplet state was demonstrated by Terenin and co-workers 22 in frozen carbohydrate solutions. However it re-quired sufficient closeness of the energy exchange partners to make their total spin a significant invariant and it may be argued that what we know about the chlorophyll monolayers in vivo does not justify this assumption.The question is obviously in need of quantitative theoretical and experimental study. The type of energy transfer considered so far corresponds to fig. l(a)-the Forster-Heller-Marcus mechanism of the migration of an intramolecular exciton, without separation of charges. Wannier's mechanism (fig. 1 (b)) may contribute to this migration. It may be asked more importantly whether the formation and transfer of intermolecdar excitons transfer (fig. 1 (c)) involving separation of charges can and does occur in the chloroplasts. Mechanisms of this type were postulated by Arnold 17 to account for the light-induced electron trapping in dried chloroplast layers (and by Commoner et aZ.23 and Calvin et aZ.7.2) to account for the occurrence of unpaired spins in illuminated chloroplast preparations Cre-vealed by paramagnetic resonance).However one difficulty opposes itself to the postulate that light absorption by chlorophyll in the chloroplast leads to electron transfer into a " conductance band "-in other words into a state in which the electron is separated from the hole (the two forming an intermolecular exciton), so loosely bound as to be dissociable by thermal motion or by a small applied external electric field. This dif€iculty lies in the fact that the absorption band of chlorophyll in vivo is very similar to that in vitro ; this suggests that the excitation leads to the same essentially intramolecular excited state in which electronic excitation is still coupled to intramolecular vibrations.Resonance energy transfer (simultaneous hole-electron transfer) can occur as an " afterthought " after such an absorption act because of the resonance between the excitation states of two chlorophyll molecules. Similar delayed transfer of the electron without the hole, i.e. conversion of an intramolecular into an intermolecular exciton (as in fig. lc), could occur only if the energy level of the second is nearly the same as (or below that of) the first-which appears unlikely. Since the quantum yield of the observed creation of trapped electrons and of free electron spins appears to be very low it may be suggested that these follow not normal but exceptional absorption acts e.g.such occurring in chlorophyll molecules immediately adjacent to impurities which can serve as electron traps. Furthermore effects observed with dried chloroplasts may well be due in part or entirely to chlorophyll aggregates formed as a result of drying. Brody 25 has observed that illumination of crystalline chlorophyll powder does lead to abundant production of free electron spins. Franck suggested that free electrons may be viable in the water-free lipoid-protected parts of the chlorophyll layers. However Franck's concept of the mechanism of photosynthesis 15 does not require the occurrence of free electrons ; he merely admits the possibility of their occurrence on the basis of Arnold's findings.Pending experimental proof of the relevancy of the findings of para-magnetic resonance of photoconductivity and of delayed light emission stimulated by heating to the main reaction sequence in photosynthesis this author is inclined to consider these phenomena as associated with minor side-processes. Calvin and co-workers 7.24 consider the evidence of the presence of free elec-trons as significant because they think that photochemical separation of charges, followed by electron migration across the lamella (and hole migration in the opposite direction) can produce effective separation of the primary oxidation products from the primary reduction products in photosynthesis. (This author has repeatedly pointed out that this separation is the real crux of photosynthesis ; in non-living photochemical processes high energy intermediates often can b 170 PRIMARY PROCESSES IN PHOTOSYNTHESIS produced with a good quantum yield but they tend to disappear by recombination before they can be effectively “salvaged” and light energy stored as chemical energy.) It must be realized however that in contrast to the “ solar battery ”, the active pigment layer in chloroplasts probably is only one molecular layer thick.To separate the oxidation product from the reduction product all that is needed is for the two products to be produced on two sides of this monomolecular layer. For example one could envisage the excited chlorophyll molecule giving an electron (or an H-atom) away into the aqueous phase at one of its ends and recovering it at the other end.In this way the two products could be drained into alternate interstices without the need for more electron conductance than that made possible by the aromatic structure of the chlorin system. Electron and hole migration in the plane of the lamella is a different matter. One could conceive of this motion as helping to bring these two products to two different enzymatic reaction sites in the layer-one suitable to initiate the reduction of an appropriate intermediate oxidation-reduction couple (such as TPN-TPNHz), the other suitable to initiate the generation of free oxygen. This possibility, different from that envisaged by Calvin and co-workers should be kept in mind; however it too is made unlikely by the above-mentioned spectroscopic evidence. We thus suggest as a plausible working hypothesis that migration of an intra-molecular exciton is the only significant type of energy migration in the chloroplast, and that its usefulness consists in bringing excitation energy close to the photo-enzymatic centre located in each or in each few macromolecular “ grana ” in the chloroplasts.This picture will need to be developed in more detail by specifying the location and function of the two (or several) different types of chlorophyll as well as of the accessory pigments particularly in view of Emerson’s observations 26 suggesting distinct photochemical functions of the several pigments including the chlorophylls a and b. With phycobilins it is likely that these pigments occupy their own granular macromolecules which are incorporated into the lamellae as would be components of a “mixed crystal”.With carotenoids (other than the photo-synthetically highly efficient fucoxanthol of brown algae) it seems more likely that these pigments are distributed in a different and as yet unknown way; if Platt’s picture 27 of carotenoids as “ electron pipelines ” between excited chloro-phyll molecules and electron acceptors (or donors) is correct some carotenoid molecules must be associated with the enzymatic “ reaction centres ”-in fact, their presence may conceivably defme these centres. 5. PHOTOCHEMICAL FUNCTION OF CHLOROPHYLL There are other chemical findings which require fitting into the above-presented “ photophysical ” picture. These are findings by Krasnovsky and co-workers,28 Rabinowitch and Weiss 29 and Bannister 30 of the capacity of chlorophyll for reversible photochemical reduction and oxidation suggesting that chlorophyll sensitizes photosynthesis by undergoing one or both of these reversible reactions.From the studies of the “ Krasnovsky reaction ”-reversible photochemical reduction of chlorophyll by ascorbic acid-and of chlorophyll-sensitized reductions of various oxidants by the same reductant (Evstigneev and Gavrilova,31 Bannister30) it appears that in vitro chlorophyll is first reduced to an unstable product-probably a free radical-which can rapidly reduce such relatively un-willing oxidants as safranin T or riboflavin. If not immediately re-oxidized this active intermediate is transformed (probably by dismutation) into the more stable non-radical fully reduced form which is pink for chlorophyll a (“ eosinophyll ”).Observation of “ difference spectra ” of Chlorella cells (difference between the absorption spectra of dark and illuminated cells) (Coleman and Rabinowitch 32) suggests-but does not yet prove-that this form of reduced chlorophyll ma E. RABINOWITCH 171 accumulate in cells (up to 0.3 % of total chlorophyll) when photosynthesis be-comes light-saturated. Since 0.3 % - 1/300 this finding further suggests that in photosynthesis the chlorophyll molecules directly associated with the " reaction centres " and therefore serving as " energy sinks " may become reduced (probably only to the unstable radical state as long as no light saturation occurs ; and to the valence-saturated eosinophyll when the limiting enzymatic reaction becomes unable to keep step with light absorption and light saturation ensues).This picture of the mechanism of the photophysical and primary photochemical stages in photosynthesis obviously is highly speculative and may easily prove wrong in parts or in toto. It seemed however a temptation to attempt to develop such a picture from the numerous and often unconnected data accumulated by recent research in our own and other laboratories. The experimental work in part utilized in this paper was carried out in the Photosynthesis Laboratory of the Botany Department at the University of Illinois, by E. E. Jacobs S . S . Brody M. Brody P. Latimer and T. T. Bannister with the assistance of the Office of Naval Research. 1 see for example Halbleiterproblem vol.I article by Volz and vol. IV articles by 2 Rabinowitch Ann. Rev. Plant Physiology 1953 3 229. 3 Wolken J. Gen. Physiol. 1954 37 111. 4 Thomas Minnaert and Elberts Acta Botanica Nkerlandica 1956 5 315. 5 Hanson Rec. trav. Botan. Nierland. 1939 36 183. 6 Jacobs Holt Kromhout and Rabinowitch Arch. Biochem. Biophys. 1957,72,495. 7 Bradley and Calvin Proc. Nat. Acad. Sci. 1956 42 710. Tollin Sogo and Calvin, 8 J. C. Goedheer Thesis (University of Utrecht 1957). 9 Frey-Wyssling and Steinmann Viertet'jahresshr. naturforsch. Ges. Ziirich 1953 98, 20. Steinmann Expt. Cell. Res. 1952 3 367; Experentia VIII 1952 8 300. Haken and by Schottky. J. Chin?. physique 1958 55 919. Calvin Sogo Tollin Kearns this Discussion. 10 L. M. N.Duysens personal communication. 11 Krasnovsky and Brin Compt. rend. (Doklady) Acad. Sci. U.S.S.R. 1948 62 163. Krasnovsky and Kosobutskaya Compt. rend. (Doklady) Acad. Sci. U.S.S. R. 1953, 91 343. Krasnovsky Kosobutskaya and Voynovskaya Compt. rend. (Doklady) Acad. Sci. U.S.S.R. 1953 92 1201. 12 Smith Kupke Loeffler Benitez Ahrne Giese in Research in Photosynthesis (Inter-science Publishers N.Y. 1957) p. 464. K. Shibata J. Biochem. (Japan) 1957 44, 147. 13 Brody and Rabinowitch Science 1957 125 555. 14 Latimer Bannister and Rabinowitch Science 1956 124 585. 15 Brody Science 1958 128 838. 16 Franck in Handbuch der Pflanzenphysiologie in press. Brugger and Franck Arch. 17 Arnold and Sherwood Proc. Nat. Acad. Sci. 1957 43 105. 18 Platt in Radiation Biology vol.111 (McGraw-Hill N.Y. 1956) p. 71. 19 Gaffron and Wohl Naturwiss 1936 24 81. 20 S. S. Brody and M. Brody unpublished. 21 Franck and Teller J. Chent. Physics 1938 6 861. 22 Terenin and Ermolaev Compt. Rend. (Doklady) Acad. Sci. U.S.S.R. 1952 85 547. Ermolaev News (Irvestiya) Acad. Sci. U.S.S.R. ser. fys. 1956 20 514. Ermolaev and Terenin J. Chin?. physique 1958 55 698. Commoner Heise and Townsend Proc. Nut. Acad. Sci. 1956 42 71. Commoner Heise Lippincott, Norberg Passonneau and Townsend Science 1957 126 57. Biochem. Biophys. 1958 75 465. Franck Proc. Nut. Acad. Sci. 1958 44 941. 23 Commoner Townsend and Pake Nature 1954 174 689. 24 Calvin and Sogo Science 1957 125 499. 25 S. S. Brody unpublished. 26 Emerson Chalmers and Cederstrand Proc. Nut. Acad. Sci.1956,43 133. Emerson 27 Platt Science 1958 129 372. and Chaimers Phycological Bull. 1958 11 51 172 PRIMARY PROCESSES I N PHOTOSYNTHESIS 28 Krasnovsky Compt. rend. (Doklady) Acad. Sci. U.S.S.R. 1948,60,421; this and sub-sequent papers (up to 1953) by A. A. Krasnovsky V. B. Evstignnev and co-workers are available in translation by E. Rabinowitch AEC-tr-2156 Office of Technical Services Dept. of Commerce Washington 25 D.C. More recent papers * Compt. rend. (Doklady) Acad. Sci. U.S.S.R. 1954 95 841 ; 1954 96 1025 1201 1219; 1954 98 1017; 1954 100 131; 1955 103 97 283; 1955 104 440 882; 1956 108 507; 1957 112 911; 1957 114 1066; also Biokhiiniya 1953, 18 240; 1956 21 126; News (Izvestia) Acad. Sci. U.S.S.R. Ser. Biol., 1955,122 ; Fizyol. rastenyi 1957,4,124 ; Biofizyka 1956,1 120 ; Zhurnal fyz. khimi, 1956 30 968. 29 Rabinowitch and Weiss Proc. Roy. SOC. A 1937 162 251. 30 T. T. Bannister Thesis (Univ. of Illinois 1958) ; Proc. Endicott House Conference on Photochemical Storage of Energy (Sept. 1957) in press. 31 Evstigneev and Gavrilova Compt. rend. (Dokludy) Acad. Sci. U.S.S.R. 1953 92, 381 ; 1954,95,841; 1954,96,1201; 1955,100,131 ; 1955,103,97 ; 1957,114,1066. 32 Rabinowitch in Proc. Endicott House Conference on Photochemical Storage of Energy (Sept. 1957) in press. Coleman Holt and Rabinowitch in Research in Photo-synthesis (Interscience Publishers N.Y. 1957) p. 68. Coleman and Rabinowitch, J. Physic. Chem. 1959 63 30. * Many of these available in translations by E. Milner from the Crerar Library, Chicago

ISSN:0366-9033    

DOI:10.1039/DF9592700161

出版商:RSC    

年代:1959

数据来源: RSC