Magneto Optical Rotation Spectroscopy and its Application to Studies of Biological Molecules BY VICTOR E. SHASHOUA Dept. of Biology Massachusetts Institute of Technology Cambridge, Massachusetts Received 3rd November 1969 The general features and method of measurement of magneto opical rotation (MOR) spectra are described. A number of structure-property relationships are derived from studies of the MOR spectra of haemoglobin cytochrome c and chlorophyll a and b. The results suggest that in special cases conformational changes can came indirect interactions with the chromophoric groups of these biological molecules to give changes in their MOR spectra. Magneto optical rotation (MOR) spectroscopy is based on Faraday’s discovery in 1846 that any molecule will rotate the plane of polarized light when a magnetic field is applied parallel to the light beam.This phenomenon has been the subject of many investigations at the sodium D-line frequency.2 Cotton and Scherer were first to carry out dispersion measurements at the absorption region of the visible spectrum of cobaltous chloride solutions. This observation suggested the possibility that the Faraday effect might be useful for extending the optical rotatory dispersion (ORD) technique with its limitation to naturally optically active compounds to the study of the stereochemistry of non-optically active molecules. Ill IV v l + - MOR MCD + ABS FIG. 1.-General types of MOR and MCD spectra. The initital work was carried out with magnetic fields of up to 18 000 G.5 The results showed that MOR spectra could be classified into five types.Fig. 1 illustrates the dispersion features of the magnetic rotation for the five types of MOR spectra observed at the absorption band regions of the molecules. In addition the figure depicts the corresponding magnetic circular dichroism (MCD) curves derived from MCD measurements on CoC1 and FeCl by Schooley Bunnenberg and DjerassL6 Types I and I1 MOR spectra have positive and negative sigmoid curves respectively, with inflection points coincident with the absorption maxima. Types I11 and IV 6 62 M O R SPECTROSCOPY -B I 0 LO G I CA L MOLECULES MOR spectra have negative and positive magnetic rotation peaks at the position of the absorption band maxima. Type V shows no anomalous dispersion features and may be positive or negative depending upon the sign and magnitude of the magnetic rotation for neighbouring absorption bands.The apparent similarity of the MOR spectral types to those obtained in ORD measurements however does not lead to any simple correlation of the sign and shape of the MOR spectrum with stereo-chemical features of molecules. Experimental investigations in a number of labora-tories 8-11 and the theoretical studies of Buckinghani and Stephens suggest that MOR and MCD spectroscopy as analytical techniques are applicable to a different variety of problems than those which can be studied by ORD and CD methods. These include (1) assignments of the positions of excited states in complex spectra 8-1 (2) the determination of magnetic moments of excited states of molecules 9* lo and (3) the determination of the polarization of magnetic moments of excited states.8 In addition there are a number of interesting structure property correlations which can be deduced from studies of the MOR spectra of certain biologically active mole-cules.These experimental investigations are reviewed in this paper as examples of special cases where conformational changes might cause an indirect influence on the MOR spectra. EXPERIMENTAL M E T H O D OF MEASUREMENT Fig. 2 shows a diagram of the instrument l 2 used for measuring the MOR spectra. The equipment is designed to measure the magnetic rotation induced by a 5000 G field as a function of wavelength with an angular sensitivity of ~0.001" and ~k0.003" at the visible and u.-v.spectral regions respectively. In the operation of the instrument a plane polarized light beam is rotated by an angle (@+a) in the first solenoid where 8 is the angle of rotation POLAR IZER SOLUTION MAGNET I SOLVENT MAGNET II MAG N E Ti: FIELD El YAGNETIC FIELD PHOTO -MULTIPLIER FIG. 2.-Diagram of the MOR spectropolarimeter. Thc operation of the i nstrumcnt is illustrated in the inset 8 solvent+sample cell rotation; a solute rotation and 4 modulation angle in the Faraday coil. by the solvent plus sample cell and o! is the rotation of the solute. The light beam then passes through a second solenoid with a magnetic field polarized in the opposite direction. This contains a sample cell with pure solvent to give a rotation of (-0). The net rotation of the polarized light after passing through the two magnetic fields is CI the solute rotation.The light beam then passes through a third a.c. magnetic field which modulates the light beam by using the induced Faraday rotation in a water sample 20 c ni long. This provide V l C l O K E . SHASHOUA 63 the carrier frequency for activating the angle sensing and recording sections of the apparatus. The light source was a 150 W Xe arc lamp. A Cay model 14 double monochromator l3 provided the spectral purity of wavelength selection with a stray light level of less than 1 part in lo6. The sample temperature was maintained to within 0.1"C. Under the experimental conditions used the light beam was along the north-south direction in the first magnetic field. Measurement of a clock-wise rotation of the plane of polarized light for observations opposite to the direction of travel of the light beam was designated as a positive rotation.This is in the same sense as the Faraday rotation for a pure solvent outside its absorption band region. The specific magnetic rotation [a], is defined by where 8 is the angle of rotation in degrees I the path length in dm c the concentration in g/ml and H i s the magnetic field in G. = 10 000 e l m MATERIALS The bovine haemoglobin were commercial grade samples from Sigma Chemical Company, St. Louis Missouri. The cytochrome c samples were obtained from Dr. R. W. Estabrook of the University of Pennsylvania. The chlorophyll and methyl pheophorbide a samples were obtained from Dr. M. Calvin and J.Anderson. RESULTS AND DISCUSSION MOR SPECTRA OF HAEMOGLOBIN A N D METHAEMOGLOBIN The haemoglobin chromophore is an octahedral complex with four nitrogen ligands provided by the planar porphyrin nucleus a globin at the fifth position and various substituent ligands at the sixth position about the central iron atom. Table TABLE 1 .-MOR SPECTRAL DATA FOR HAEMOGLOBIN pH = 6.8 10°C magnetic absorpt. MOR data P) moment maxima Il/nm [=Is 12/nm [akp B.M.(d) nm 554 550 45 585 150 4.9 540 538 1 00 574 510 0 540 530 210 0 572 570 650 542 535 118 577 576 785 no. unpaired electrons 4 0 0 0 (a) The [aIsp values of the MOR data are the sum of the positive and negative components of the type I11 MOR spectra as illustrated by A in fig. 5 ; (b) commercial grade bovine haemoglobin ; (c) horsc haemoglobin data ; (d) data obtained from ref.(15). 1 and fig. 3 show the MOR and ORD spectral data obtained for a number of ferro-haem~globins,'~ with HzO 0 and CO as the ligands. The natural optical rotations of these molecules are quite small for the spectral regions 450-650 nm whereas the MOR data show substantial magnetic rotations with dispersion features characteristic of a type 111 spectrum. The magnitude of the specific magnetic rotation at the 570 nm band of bovine ferrohaemoglobin increases about four fold from 150 to 650 for replacement H20 with CO as the ligand. As shown in table 1 there seems to be no relationship between the specific magnetic rotation data to paramagnetism of the samples. However a large magnetic rotation is obtained for substituents with a large ligand field.I n addition the magnitude of the specific magnetic rotatio 64 M O K S I’ I C 1’KOS CO Y Y - B 1 0 LOG 1 C A L MOLECULES *0° I----- 7 0 -200 0 -200 B -U -400 0 -200 1.0 0 pjfzj Hb-CO 0 500 600 wavelength nm FIG. 3.-MOR and ORD spectra of bovine ferrohaemoglobins with HzO O2 and CO ligands. seems to change with the source of the oxyferrohaemoglobin. The magnetic rotation for horse oxyferrohaemoglobin is 50 % higher than for bovine oxyferrohaemoglobin at the 576 nm band; however at the 535 nm region the magnetic rotation is about the same for the two proteins. This may be indicative of a ligand field change at the chromophore of the two proteins. Table 2 summarizes the results for methaemoglobin (ferrihaemoglobin) and its derivatives.In these molecules the four-absorption-band system characteristic TABLE 2.-MOR SPECTRAL DATA FOR METHAEMOGLOBIN pH 6.8 10°C band I band I1 band I11 band IV magnetic unpaired ligand Al/nm [a] sp 12/nm [a] sp 13/nm [m] sp A4/nm [a] sp moment (a) electrons 630 50 567 36 540 20 500 28 5.20 (4) F 606 95 - - 550 30 485 38 5.92 4 HzO OH@) 607 32 575 95 538 38 - - 4.47 4 CN - - 565 470 540 129 - - 2.5 1 (0) The magnetic moment data were obtained from ref. (15) (16); (b) this was studied at pH 10. of the high spin state of methaemoglobin with water as the ligaiid changes to the two absorption band system of methaemoglobin cyanide with a low spin state. The ORD spectra l4 of these molecules for the 450-650 nm region are small and show no substantial changes for various substituents.The MOR spectra however show large magnetic rotation changes. A comparison of A as defined in fig. 5 for th VICTOR E . SHASHOUA 65 band I11 system (540 nm) shows an increase in the specific magnetic rotation from 20 for H,O as the lilrand to 129 for cyanide as the limand. Similar changes occur for the band I1 system at the 570nm region i.e. from 36 to 470. All the MOR spectra are of type 111. A large magnetic rotation appears to be characteristic of methaemoglobin cyanide (low spin state) while small magnetic rotations are observed for the high spin haemoglobin compounds with H20 OH and F as the ligands. These results suggest a correlation of the magnitude of magnetic rotation with the spin state of the compounds.MOR SPECTRA OF CYTOCHROME C The MOR spectrum of cytochrome c is very sensitive to the oxidation state of the m01ecule.l~ The specific magnetic rotation changes from - 8000 to - 150 at 546 nm. The type I11 magnetic rotation at the 549 nm region (see fig. 4) of the spectrum was used for the study of the kinetics of oxidation and reduction of the 0 0 . 2 0 -so2 0 d 2 .- -.04 Y -.O 6 .-o 8 - - I 0 5 0 0 5 5 0 5 0 0 5 5 0 wavelength nm FIG. 4.-MOR spectra of ferrocytochrome c derived from yeast and pigeon breast. TABLE 3.-MOR SPECTRAL DATA-CYTOCHROME C pH = 7 pH = 10 source A B A B yeast 1.77 0.22 1.70 0.37 Tuna 1.75 0.21 1.80 0.21 horse H. 1.79 0.23 1.71 0.15 pigeon B. 1.75 0.23 1.71 0.15 A is the ratio of optical densities at 550 to 520nm.B is the A MOR divided by the optical density. molecule.ls One interesting feature of the MOR spectrum of cytochrome c was observed in a study of samples derived from different sources.19 Fig. 4 compares the MOR results for yeast and pigeon breast ferrocytochrome c at pH 10 for the a and p band absorption regions. The ORD measurements are quite small for this region. Table 3 presents an analysis of the 549 nm MOR rotation for different samples at pH 7.0 and pH 10. All the samples were first converted to the fully reduced state and the spectra were determined in the presence of reducing agent s3-66 MOR SPECTROSCOPY-BIOLOGICAL MOLECULES (formamidine sulphinic acid 20) under a nitrogen atmosphere. The ratio 1.7 to 1.8 of the optical densities of the a and j3 bands was used as a criterion of the fully reduced state of ferrocytochrome c.As shown in table 3 the magnetic rotation per optical density unit (B in table 3) varied for the different samples when the measurements were carried out at pH 10 near the isoelectric point of ferrocytochrome c ; but no change occurred at pH 7.0. Thus yeast cytochrome c had over twice the magnetic rotation of pigeon breast cytochrome c. There are a number of explanations which might account for the observed data. Since all the molecules have the same chromo-phoric group with a ferrous atom at the centre of an octahedral complex the changes in the observed magnetic rotation cannot be due to a major structural change. One possibility similar to the data for ferrohaemoglobin is the use of different ligands in the various cytochrome c molecules.Another alternative is that the known different polypeptide sequences near the chromophoric group can provide a change in the effective pH around the porphyrin nucleus. It is known that pH changes can vastly influence the MOR spectrum of cytochrome c,l* thus it would 500 510 520 530 540 550 560 570 wavelength nm 1 ~ 1 1 1 1 1 1 1 1 10 20 . 30 40 50 60 70 80 temp. "C FIG. 5.-Teniperature effects on the MOR spectrum of ferro and ferricytochrome c. Fig. 5~ shows the spectra for ferrocytochrome c at 16 and 71.5"C. Fig. 5~ is a plot of A for a 0.05 % solution for ferrocytochrome c and A at 0.18 % solution for ferricytochrome c. not be surprising that such a change could result in a conformation change and that this would more easily occur at the isoelectric point of the molecule.This example illustrates the possibility that structural changes can influence the observed MOR spectrum; Another example of indirect stereochemical effects on MOR spectra is illustrated in fig. 5. A study of the magnetic rotation of ferrocytochrorne c. in the presence o 67 METHYL PHEOPHORBIDE a in CCL,, -48-I ' / / I 1 I ;ABSORPTION -\ ' \ -,AT 0.0031 O/o 1 I I I \ \ \ L A - __A_- -r'Lt-- ' f? I 2 3 77 X Y FIG. n I 2 X a T Y .- s .c - a 1.0 FIG. -1.0-- 2-0 METHYL PHEOPHORBIDE a in NMP 350 400 450 500 550 600 650 700 750 wavelength nm 7.-MOR and ORD spectra of methylpheophorbide a in N-methylpyrrolidone (NMP).,/'.\ ABSORPTION ' \\ AT 0.002 O/o _-___. T A- _ _ _ _ _ _ _ _ - - _--- I_ I . 300 350 400 450 500 550 600 650 700 wavelength nm FIG. 8.- MOR and ORD spectra of chlorophyll a 68 MOR SPECTROSCOPY-BIOLOGICAL MOLECULES reducing agent as a function of temperature showed that the magnetic rotation can decrease at elevated temperatures. Thus a plot of A (as defined in fig. 5) as a function of T shows a progressive decrease from 15 up to 71.5"C. Moreover upon cooling the rotation returns to approximately its original value indicating that no permanent configurational change had occurred. This type of temperature effect was not observed for the oxidized ferricytochrome c. It is possible that a change in the ligand field around the octahedral complex of the porphyrin nucleus can occur in ferrocytochrome c but not in ferricytochrome c where the extra valence of the iron atom provides stability.The ligand field change could arise from indirect effects of temperature-induced conformational changes in the polypeptide fragments of the molecule. These could produce a decrease in the ligand field in the ferro compound but may not affect the more tightly bound ferri structure of the molecule. 0 - 1.0 - 2.0 I ) I \ -/// ABSORPTION - 1.0 I , \ /,' \,AT 0.002 Yo x. _ _ _ - ~ - _ - - --. - - - - - -I z-.-L_ '-t-wavelength nm FIG. 9.-MOR and ORD spectra of chlorophyll 6. TABLE 4.-MOR SPECTRAL DATA FOR METHYLPHEOPHORBIDE a AND CHLOROPHYLL a AND b A/nm type Ilnm type A/nm type A/nm type methylpheophorbide n chlorophyll a chlorophyll b CC14 solvent NMP solvent CCI4 solvent CC14 solvent I - - _ L - l L 67 1 I1 668 I1 665 I1 646 I1 61 5 I11 610 I11 618 111 ? 600 I11 ? 537 I 538 I 585 I 508 I11 ? 509 111 ? 460 IV 417 IV 41 5 IV 432 IV NMP N-methyl pyrrolidone 0 MOR SPECTRA OF METHYLPHEOPHORBIDE a AND CHLOROPHYLL a AND b Fig.6-9 and table 4 summarize the MOR spectral data for methylpheophorbide a and chlorophyll a and b. The magnetic rotations are of the same order of magnitude as the ORD data. At the dilute solutions studied the MOR spectral types seem to follow the same general pattern for all three compounds. Methylpheophorbide a as the chromophoric group for the chlorophylls has the most distinct features with a type I1 MOR spectrum at the 671 nm band and a type I at the 537 nm in CC14.By analogy to other studies in the porphyrins,8 these two bands should have magnetic dipole transitions polarized at right angles and in the plane of the porphyriii nucleus VICTOR E . SHASHOUA 69 Briat and Djerassi 21 have reported the MCD spectrum for niethylpheophorbide a. These seem to have different relative intensities from the observed MOR data. Such variations might arise from the known association of the molecules at higher concentra-tion.22 In fact MOR and MCD spectroscopy may be useful in studying such inter-molecular association^.^^ The results described in this paper summarize a number of structural effects in special types of molecular interactions that can be observed by MOR spectroscopy.' M. Faraday Phil. Trans. 1846 3 1. for a general review see J. R. 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