IV. PROTECTION AND SENSITIZATION PROTECTION EFFECT AND ITS SPECIFICITY IN IRRADIATED AQUEOUS SOLUTIONS BY WALTER M. DALE Christie Hospital and Hol t Radium Institute Manchester 20 Received 14th January 1952 Systems which are initially one-solute systems become multi-solute as a result of irradiation. Dissolved oxygen can be a second solute leading to reaction products like HOz and H202 which can cause chemical effects. Radiation effects in two-solute systems have been investigated on two different but complementary lines. First the estimation of gaseous end-products and secondly the estimation of a change of the initial solute. A second solute in a two-solute system acts as a competitive acceptor of free radicals thus reducing the radiation effect on the first solute i.e.protecting it. This protective effect can be used as a measure of the protective power of various solutes relative to the first solute (the indicator). The specifi- city of this effect in particular that of sulphur is demonstrated in experiments with the enzyme carboxypeptidase and with alloxazin-adenine dinucleotide as the indicators. The protective power per unit mass of protector is sometimes dependent on the con- centration of the protector. One has to assume in such cases that protector molecules can hand on energy to the indicator. In order to discuss radiation effects in multi-solute systems I start with some general considerations which form the background against which the experi- mental results have to be seen. Practically all systems which are initially one-solute systems become multi- solute solutions as a result of irradiation.A one-solute system would require the absence of dissolved oxygen and secondly that only one reaction takes place whose reaction products are not further capable of reacting with free radicals resulting from the decomposition of water i.e. the reaction product has to be inert towards radiation. Some solutions of inorganic solutes capable of being reduced or oxidized come nearest to this ideal one-solute system though there is evidence that reduction and oxidation can take place simultaneously and therefore the stipulation of an inert reaction product is not strictly fulfilled. Dissolved oxygen as mentioned before acts as a second solute which by secondary reactions causes the formation of hydrogen peroxide or of the radical H02.These then lead in some cases to an enhancement of the radiation effect. There are however examples-for instance the inactivation of the enzyme carboxypeptidase and also of ribonuclease 1-which are independent of the presence of oxygen. I should like here to comment briefly on the statement often made that it is extremely difficult to remove oxygen effectively. This is quite true when the aim of the experiment is to establish an absolute quantitative relationship for reactions in presence or in absence of oxygen. It is not difficult however to reduce the oxygen tension in small volumes sufficiently to decide whether or not there is an effect of oxygen. Another point I should like to mention here is the radiation effect on solutions of methylene blue about which there seems to be some confusion in the literature.All investigators agree that bleaching occurs when the dye is exposed to ionizing radiations. This is often taken as being due 29 3 PROTECTlOF4 EFFECT AND ITS SPE<'IFICITY 294 to a reduction of the dye to its leuco-base. One has however to distinguish between the bleaching occurring in an oxygenated and in an oxygen-free solution. In oxygen-free solution the blue colour can be partially restored after irradiation when the solution is shaken with air or oxygen. In this case one deals with a predominant reduction by radiation of the methylene blue to leuco-methylene blue. In an oxygenated solution however an irreversible change of the mole- cule takes place which also appears as bleaching of the dye but the solution once bleached can never regain its colour on being shaken with air or oxygen.In other words an irreversible change has taken place which is more than a simple reductive change. Mention is now made of some difficulties with which an analysis of radiation reactions is faced when it aims at a complete insight into the primary reaction as well as into secondary reactions difficulties which become ever greater the more complex the substances and systems which are involved. This explains why many investigators have chosen the simplest solutes possible hoping to free themselves of the limitations imposed by this complexity. It is however note- worthy that even in systems in which free radicals are uniformly generated by chemical means several alternative reactions often present themselves and that the analysis cannot go further than to attribute a greater probability to some reactions than to others.The formation of free radicals by ionizing radiations introduces the additional difficulty of the discontinuity of the primary act and it is then justified to ask how far these reactions can be treated as occurring in a homogeneous system. One might be led to conclude from these remarks that no useful purpose could be served by investigating more complex systems at all but I think this would be unnecessarily pessimistic. The investigation of radiation effects has proceeded on two distinct lines. First the estimation of end products in the form of the gases H2 and C02 a line which was followed mainly by Fricke and his colleagues and secondly the deter- mination of the disappearance of an initial solute sometimes supplemented by the identification of some intermediate product into which the primary solute was changed by irradiation.Disappearance includes for a biologically active sub- stance the change from an active to an inactive molecule. Neither of the two lines of approach is without limitations. At first sight it may seem as if the first method the determination of gaseous end-products is unequivocal and will at least give an overall balance of the reaction. However uncertainty of the action of H atoms and OH radicals must sometimes exist since H atoms can dehydrogenate i.e.oxidize as well as hydrogenate and as pointed out recently by Evans and Uri,2 by Haissinsky,3 and by Weiss,4 OH radicals can oxidize as well as reduce. Furthermore carbon-carbon bonds can be broken leading to reaction products of lesser carbon content but of more stable nature. On balance solutes may by interaction with radicals catalyze the recombination of H and OH. The second line of approach followed in my laboratory and applicable to substances of more complex structure does not concern itself with the overall balance of end products and intermediate reactions but with the loss of a specific property of the solute molecule in fact for biologically active molecules for instance enzymes viruses proteins we are more interested to know the efficiency with which their specific activity is affected by radiation than the contribution they make to the total output of gaseous end products an output which according to Fricke,s is very small for proteins.In short each of the two methods exists in its own right and both are complementary. The important feature of experiments with multi-solute systems is that they offer a means of measuring to what extent a second solute added to the first is able to influence the radiation effect on the first by acting as a competitive acceptor of free radicals and so decreasing the radiation effect on the first solute i.e. WALTER M . DALE 295 protecting it. Addition of substances differing in composition then gives a picture of how their capacity to act as acceptors changes in accordance with their specific structure.Experiments of this kind were carried out by Fricke and his co- workers on solutions of simple organic solutes mainly of the aliphatic series determining the total output of C02 and H2. Without considering these experiments here in detail one can say that the reaction concentration curves of single solutes are often of a complicated nature and although one would think that simple structures of molecules should also show simple concentration reaction relationships this is by no means the case. Thus Fricke in an earlier interpretation of his concentration-reaction curves assumed that in certain cases a different chemical reaction takes place in a dilute solution from that which occurs in a more concentrated solution although in each case the solute was changed by radiation only to a minute extent.He has now also suggested that the CO2 yield dependence on concentration for the same experimental results could be interpreted in terms of chain reactions sustained by radicals formed from the solute.7 This explanation seems to be more satis- factory since it is not easy to believe that a different chemical reaction should take place simply on increasing the concentration of the solute. Such difficulties existing in one-solute systems are increased when solutes occur in multi-solute systems. Before I proceed to mention our experiments on two-solute systems I shall quote a few examples in which an increase of the radiation effect caused by the presence of a second solute is reported.There are experiments of Frickes in which an increase in reduction takes place when solutions of potassium dichromate mixed with various aliphatic acids are irradiated There are also other cases where X-rays cause reduction coupled with an increase of the effect by adding substances capable of removing oxidizing OH radicals. Stein and Day have used the reduction of oxygen-free solutions of methylene blue for X-ray dosimetry and could enhance the bleaching effect of X-rays by adding sodium benzoate.9 The same mechanism is suggested by Forssberg 10 for explaining the increase in the inactivation of catalase in presence of reduced glutathione and cysteine. One would assume then that in cases where H atoms are responsible for an observed radiation effect the addition of reducing substanccs will enhance and where OH radicals are responsible the addition of reducing substances will depress a radiation effect.This assumption however would not fit in with the dual role of reducing and oxidizing property of the OH radical mentioned earlier. In our experiments with two-solute systems 11 12 the first solute was the enzyme carboxypeptidase and the determination of its inactivation by radiation in presence and in absence of a second solute formed the basis on which the ability of various second-solutes to react with free radicals could be compared. Since there was always a decrease of the radiation effect in presence of the substances added we speak of the second solute as the protector of the enzyme the latter being called the indicator.We also have used alloxazin-adenine dinucleotide as the indicator. This dinucleotide is only the prosthetic group of the enzyme D-amino acid oxidase and therefore itself inactive but becomes active in presence of a specific protein. One can determine the chemical change of the dinucleotide after irradiation by building up the complete system dinucleotide (D) specific protein (P) and the substrate alanine (A) and measuring the oxygen consumed for oxidation of the alanine.12 The two indicators carboxypeptidase and dinucleo tide differ widely in their molecular weight 35,000 and 920 respectively but both have in common that the efficiency of radiation (ionic yield) remains constant over a wide range of initial concentrations.The following experiment (table 1) illustrates the protective effect of various concentrations of leucylglycine added to dinucleotide before irradiation. The PROTECTION EFFECT AND ITS SPECIFICITY 296 oxygen uptake increases with decreasing concentration of leucylglycine until it reaches the value of the control. Similar experiments with carboxypeptidase as indicator had results shown in table 2.13 It will be seen that the first six substances of very different molecular weights but otherwise of similar average composition have practically the same pro- tective power per microgram of protector and therefore the protective power per molecule which is proportional to the molecular weight is an additive manometer 2 3 1 4 5 6 substance TABLE I * X-ray dose = 4000 r.L == leucylglycine [ ]* = irradiated contents D f P f A [D + 10-4 mole L]* + P -1- A [D + 10-6 mole L]* 4- P 4 A [D + 10-7 mole L]* t P i- A [ D -1 10-8 mole L]* t P + A [Dj* -t P t A TABLE 2 -f mol. wt. 48 x 106 7.6 s 106 40,000 - 89 180 45 88 60 142 116 tobacco mosaic virus bushy stunt virus cryst. egg albumin denat. egg albumin alanine glucose formate H-COONa COONa oxalate COONa iVH2 thiourea C=S I I NH2 urea I I I NH2 c=o NH2 alloxan CO CO NH-CO I I NH-CO mesoxalate I I COONa I c=o I COONa * reproduced by permission of the Biochemical Journal.7 reproduced by permission of the British J . Radiology. 7 pI. 0 2 in 1st 10 min 28.6 27-1 19.3 12-8 10.0 10.7 rel. protective power per pg 30 20 17 20 39 34 320 1.5 -1 120 0.5 13 WALTER M. DALE 297 function of the molecular groups constituting this molecule. The next six sub- stances however contain special groups of atoms which form a large part of the total molecule and these have a very marked and specific effect on the protective power. Such specific effects are masked by the average uniformity of the com- position of large molecules. Of special interest is the change of protective power caused by the substitution of a sulphur atom for an oxygen atom in urea and this has led to an investigation of other sulphur-containing compounds and of sulphur itself.14 Table 3 summarizes the results.Column 1 gives the weight of protector per ml used in the experiment and column 2 the respective sulphur content. Column 3 gives the protective power ( Q p ) per microgram of protector and column 4 the protective power Qs of such amounts of protector as contain 1 microgram of sulphur in each case. One can therefore estimate from column 4 how the non-sulphur residue in any one com- pound affects the protective power of 1 microgram of sulphur contained in it taking colloidal sulphur as reference. Elemental sulphur is about as protective 3 TABLE 3 $ 1 p g protector per ml 4 QS 130 58 5.0 5.0 1.6 1-6 thiourea dimethylthiourea colloidal sulphur 2 pg sulphur per ml 2.1 1.54 1-06 (for one S) QP 55 18 110 24 110 118 sodium thiosulphate 5.3 as thiourea and sodium thiosulphate but the introduction of two methyl groups into thiourea causes a considerable decrease in protective power of the sulphur The protective power of a protector can be expressed by the quotient Q = (DP+I - Dz)/’DI - I/P where Dr is the X-ray dose required to change the indicator by 63 % in absence of protector and Dp+ I the X-ray dose required to change the indicator by 63 % in presence of protector and I and P the concentration (pglrnl) of indicator and protector respectively.Q therefore represents the proportion of the X-ray dose taken up by the protector to that taken up by the indicator for equal weight of protector and indicator.In uncomplicated cases Q is independent of the concentration of protector used. In other cases however one finds that Q decreases with increasing concentration of protector as shown in fig. 1. Such a decrease of Q can be explained by assuming that energy taken up by the protector is handed on to the indicator. It is however not to be assumed that the decrease of Q is caused by a direct action of radiation on the protector molecule. Such a direct effect would require much higher concentrations of protector than those at which the decline of the protection effect becomes evident and would lead to improbably high figures of the ionic yield when calculated on the basis of a direct mode of action.We have developed formulae based on the collision frequencies of a radical with protector and indicator molecules of given molecular weight which are valid for all cases regardless of the existence or non-existence of a handing-on effect and the values of the ratios of the probabilities of destruction of a radical by collision with a protector and an indicator molecule respectively are collected in table 4. The ratios pp/pR are a measure of the protective power per molecule of the protector for the given indicator. It will be seen that the protective power of thiourea is 10,000-fold greater than that of urea and further that a change from the indicator carboxypeptidase (C.P.) to the dinucleotide (D.N.) does not appreci- ably change the values of the protective power although these indicators are rather 1 reproduced by permission of the BritisJi J.Cnnccr. K PROTECTION EFFECT AND ITS SPECIFICITY 298 different. It seems as if it were advantageous to use indicators which do not exhibit complicated concen trat ion-radia t ion dosage relationships. Finally the protection effect also operates in systems which are not true solu- tions. This has been shown in experiments with suspensions of biological units protector thiourea sodium formate dime thy1 thiourea glucose dimethylurea egg albumin alloxan sodium mesoxalate sodium oxalate urea 0 \ 0 8 A ‘a Q e.g. bacteria and sperm of arbaciae to which various substances were added as protectors.Hollaender and his colleagues have used the survival rate of coli bacteria when irradiated in presence and in absence of alcohol SH compounds etc.,ls and Evans and his colleagues 16 used the change of fertilizing power of arbacia sperm as indicator of radiation effects. Such experiments have clearly shown the protective action of substances dissolved in the surrounding medium. The interpretation however is complicated by the comparatively enormous 2.0 x 10-3 7.5 :. 10-4 “ Changing “ Changing quotient quotient ” for for C.P. C.P. Curves points Curves are are theoretical theoretical ; points expt. expt. A dimethylurea A Jf Jf dimethylurea with with C.P. C.P. 30 30 pg/ml B B 0 glucose with glucose with C.P. C.P. 30 30 pg/ml pg/ml ucose ucose with with C.P.C.P. 90 90 pg/ml pg/ml \ 0.24 size of the particles and the existence of cell membranes acting as permeability barriers and introducing surface phenomena. If the substance does not penetrate into the interior of the particle its effect is confined to competitive action in the surrounding fluid and the surface layers of the particle and can be understood by assuming that these surfaces play an active part in the maintenance of the bio- logical activities of the cell. A full explanation of the mechanism will therefore require an analysis of permeability as well as an investigation of the question whether such surfaces can be the link between radiation and the effects observed. f reproduced by permission of the British J . Cancer. conc. i n /ig,ml.TABLE 4 f mol. wt. 76 68 104 180 88 -40,000 142 152 134 60 Q p p l p ~ with D.N. p p l p ~ with C.P. 2.1 0.74 1.9 - 0-21 - 0.21 - 4-7 3-1 0.79 0.4 2.1 x 10-2 2.4 x 10-2 - - - 299 In contrast to systems of suspended particles is the condition of gels in which a colloid forms a continuous network of micelles between which the solvent circulates. Zt is important from the biological point of view that no protective action by the gelatinous network was observed by Skoog17 and Gordon and Quastler 18 when they irradiated Auxin in agar blocks or by Day and Stein 9 in their experiments with methylene blue in gelatine gels. These systems are of biological importance as models of the inhomogeneous physical structure of the interior of cells referred to in our experiments.13 WALTER M .D A L E 1 Dainton and Holmes Nature 1950 165 266. 2 Evans and Urj Nature 1950 166 602. 3 Haissinsky and Lefort Compt. rend. 1950 230 1156; J . Chim. Phys. 1950 47 588. 4 Weiss Reunion Ann. SOC. Chim. Phys. (1951). 5 Fricke Cold Spring Harbor Symp. 1935 6 164. 6 Fricke Hart and Smith J . Chem. Physics 1938 6 229. 7 Fricke Symp. 4 (1950) Sept. 18-20 Army Medical Center Md.). 8 Fricke and Brownscornbe J . Amer. Chem. Soc. 1933 55 2358. 9 Stein and Day Nature 1950 166 146. 10 Forssberg Nature 1947 159 308. 11 Dale Brit. J. Rad. 1943 16 171. 12 Dale Biochem. J. 1942 36 80. 13 Dale Brit. J. Rad. 1947 Suppl. 1 46. 14 Dale Davies and Meredith Brit.J . Cancer 1949 3 31. 15 Burnett Stapleton Morse and Hollaender Proc. SOC. Expt. Biol. Med. 1951,77 636. 16 Evans Slaughter Little and Failla Radiol. 1942 39 663. 17 Skoog J. Cell. Cohp. PJiysiol. 1935 7 227. 18 Gordon and Quastler (private communication). IV. PROTECTION AND SENSITIZATION PROTECTION EFFECT AND ITS SPECIFICITY IN IRRADIATED AQUEOUS SOLUTIONS BY WALTER M. DALE Christie Hospital and Hol t Radium Institute Manchester 20 Received 14th January 1952 Systems which are initially one-solute systems become multi-solute as a result of irradiation. Dissolved oxygen can be a second solute leading to reaction products like HOz and H202 which can cause chemical effects. Radiation effects in two-solute systems have been investigated on two different but complementary lines.First the estimation of gaseous end-products and secondly the estimation of a change of the initial solute. A second solute in a two-solute system acts as a competitive acceptor of free radicals thus reducing the radiation effect on the first solute i.e. protecting it. This protective effect can be used as a measure of the protective power of various solutes relative to the first solute (the indicator). The specifi-city of this effect in particular that of sulphur is demonstrated in experiments with the enzyme carboxypeptidase and with alloxazin-adenine dinucleotide as the indicators. The protective power per unit mass of protector is sometimes dependent on the con-centration of the protector. One has to assume in such cases that protector molecules can hand on energy to the indicator.In order to discuss radiation effects in multi-solute systems I start with some general considerations which form the background against which the experi-mental results have to be seen. Practically all systems which are initially one-solute systems become multi-solute solutions as a result of irradiation. A one-solute system would require the absence of dissolved oxygen and secondly that only one reaction takes place whose reaction products are not further capable of reacting with free radicals resulting from the decomposition of water i.e. the reaction product has to be inert towards radiation. Some solutions of inorganic solutes capable of being reduced or oxidized come nearest to this ideal one-solute system though there is evidence that reduction and oxidation can take place simultaneously and therefore the stipulation of an inert reaction product is not strictly fulfilled.Dissolved oxygen as mentioned before acts as a second solute which by secondary reactions causes the formation of hydrogen peroxide or of the radical H02. These then lead in some cases to an enhancement of the radiation effect. There are however examples-for instance the inactivation of the enzyme carboxypeptidase and also of ribonuclease 1-which are independent of the presence of oxygen. I should like here to comment briefly on the statement often made that it is extremely difficult to remove oxygen effectively. This is quite true when the aim of the experiment is to establish an absolute quantitative relationship for reactions in presence or in absence of oxygen.It is not difficult however to reduce the oxygen tension in small volumes sufficiently to decide whether or not there is an effect of oxygen. Another point I should like to mention here is the radiation effect on solutions of methylene blue about which there seems to be some confusion in the literature. All investigators agree that bleaching occurs when the dye is exposed to ionizing radiations. This is often taken as being due 29 294 PROTECTlOF4 EFFECT AND ITS SPE<'IFICITY to a reduction of the dye to its leuco-base. One has however to distinguish between the bleaching occurring in an oxygenated and in an oxygen-free solution. In oxygen-free solution the blue colour can be partially restored after irradiation when the solution is shaken with air or oxygen.In this case one deals with a predominant reduction by radiation of the methylene blue to leuco-methylene blue. In an oxygenated solution however an irreversible change of the mole-cule takes place which also appears as bleaching of the dye but the solution once bleached can never regain its colour on being shaken with air or oxygen. In other words an irreversible change has taken place which is more than a simple reductive change. Mention is now made of some difficulties with which an analysis of radiation reactions is faced when it aims at a complete insight into the primary reaction as well as into secondary reactions difficulties which become ever greater the more complex the substances and systems which are involved.This explains why many investigators have chosen the simplest solutes possible hoping to free themselves of the limitations imposed by this complexity. It is however note-worthy that even in systems in which free radicals are uniformly generated by chemical means several alternative reactions often present themselves and that the analysis cannot go further than to attribute a greater probability to some reactions than to others. The formation of free radicals by ionizing radiations introduces the additional difficulty of the discontinuity of the primary act and it is then justified to ask how far these reactions can be treated as occurring in a homogeneous system. One might be led to conclude from these remarks that no useful purpose could be served by investigating more complex systems at all but I think this would be unnecessarily pessimistic.The investigation of radiation effects has proceeded on two distinct lines. First the estimation of end products in the form of the gases H2 and C02 a line which was followed mainly by Fricke and his colleagues and secondly the deter-mination of the disappearance of an initial solute sometimes supplemented by the identification of some intermediate product into which the primary solute was changed by irradiation. Disappearance includes for a biologically active sub-stance the change from an active to an inactive molecule. At first sight it may seem as if the first method the determination of gaseous end-products is unequivocal and will at least give an overall balance of the reaction.However, uncertainty of the action of H atoms and OH radicals must sometimes exist since H atoms can dehydrogenate i.e. oxidize as well as hydrogenate and as pointed out recently by Evans and Uri,2 by Haissinsky,3 and by Weiss,4 OH radicals can oxidize as well as reduce. Furthermore carbon-carbon bonds can be broken, leading to reaction products of lesser carbon content but of more stable nature. On balance solutes may by interaction with radicals catalyze the recombination of H and OH. The second line of approach followed in my laboratory and applicable to substances of more complex structure does not concern itself with the overall balance of end products and intermediate reactions but with the loss of a specific property of the solute molecule in fact for biologically active molecules for instance enzymes viruses proteins we are more interested to know the efficiency with which their specific activity is affected by radiation than the contribution they make to the total output of gaseous end products an output which according to Fricke,s is very small for proteins.In short each of the two methods exists in its own right and both are complementary. The important feature of experiments with multi-solute systems is that they offer a means of measuring to what extent a second solute added to the first is able to influence the radiation effect on the first by acting as a competitive acceptor of free radicals and so decreasing the radiation effect on the first solute i.e. Neither of the two lines of approach is without limitations WALTER M .DALE 295 protecting it. Addition of substances differing in composition then gives a picture of how their capacity to act as acceptors changes in accordance with their specific structure. Experiments of this kind were carried out by Fricke and his co-workers on solutions of simple organic solutes mainly of the aliphatic series, determining the total output of C02 and H2. Without considering these experiments here in detail one can say that the reaction concentration curves of single solutes are often of a complicated nature, and although one would think that simple structures of molecules should also show simple concentration reaction relationships this is by no means the case. Thus Fricke in an earlier interpretation of his concentration-reaction curves, assumed that in certain cases a different chemical reaction takes place in a dilute solution from that which occurs in a more concentrated solution although in each case the solute was changed by radiation only to a minute extent.He has now also suggested that the CO2 yield dependence on concentration for the same experimental results could be interpreted in terms of chain reactions sustained by radicals formed from the solute.7 This explanation seems to be more satis-factory since it is not easy to believe that a different chemical reaction should take place simply on increasing the concentration of the solute. Such difficulties existing in one-solute systems are increased when solutes occur in multi-solute systems.Before I proceed to mention our experiments on two-solute systems I shall quote a few examples in which an increase of the radiation effect caused by the presence of a second solute is reported. There are experiments of Frickes in which an increase in reduction takes place when solutions of potassium dichromate mixed with various aliphatic acids are irradiated There are also other cases where X-rays cause reduction coupled with an increase of the effect by adding substances capable of removing oxidizing OH radicals. Stein and Day have used the reduction of oxygen-free solutions of methylene blue for X-ray dosimetry and could enhance the bleaching effect of X-rays by adding sodium benzoate.9 The same mechanism is suggested by Forssberg 10 for explaining the increase in the inactivation of catalase in presence of reduced glutathione and cysteine.One would assume then that in cases where H atoms are responsible for an observed radiation effect the addition of reducing substanccs will enhance and where OH radicals are responsible the addition of reducing substances will depress a radiation effect. This assumption however, would not fit in with the dual role of reducing and oxidizing property of the OH radical mentioned earlier. In our experiments with two-solute systems 11 12 the first solute was the enzyme carboxypeptidase and the determination of its inactivation by radiation in presence and in absence of a second solute formed the basis on which the ability of various second-solutes to react with free radicals could be compared.Since there was always a decrease of the radiation effect in presence of the substances added we speak of the second solute as the protector of the enzyme the latter being called the indicator. We also have used alloxazin-adenine dinucleotide as the indicator. This dinucleotide is only the prosthetic group of the enzyme D-amino acid oxidase and therefore itself inactive but becomes active in presence of a specific protein. One can determine the chemical change of the dinucleotide after irradiation by building up the complete system dinucleotide (D) specific protein (P) and the substrate alanine (A) and measuring the oxygen consumed for oxidation of the alanine.12 The two indicators carboxypeptidase and dinucleo tide differ widely in their molecular weight 35,000 and 920 respectively but both have in common that the efficiency of radiation (ionic yield) remains constant over a wide range of initial concentrations.The following experiment (table 1) illustrates the protective effect of various concentrations of leucylglycine added to dinucleotide before irradiation. Th 296 PROTECTION EFFECT AND ITS SPECIFICITY oxygen uptake increases with decreasing concentration of leucylglycine until it reaches the value of the control. Similar experiments with carboxypeptidase as indicator had results shown in table 2.13 It will be seen that the first six substances of very different molecular weights but otherwise of similar average composition have practically the same pro-tective power per microgram of protector and therefore the protective power per molecule which is proportional to the molecular weight is an additive TABLE I * X-ray dose = 4000 r.L == leucylglycine [ ]* = irradiated manometer contents pI. 0 2 in 1st 10 min 1 D f P f A 28.6 2 27-1 3 [D + 10-6 mole L]* 4- P 4 A 19.3 4 [D + 10-7 mole L]* t P i- A 12-8 6 [Dj* -t P t A 10.7 [D + 10-4 mole L]* + P -1- A 5 [ D -1 10-8 mole L]* t P + A 10.0 TABLE 2 -f rel. protective power per pg substance mol. wt. tobacco mosaic virus 48 x 106 30 bushy stunt virus 7.6 s 106 20 cryst. egg albumin 40,000 17 20 denat. egg albumin -alanine 89 39 glucose 180 34 formate H-COONa 45 320 oxalate I 88 1.5 COONa COONa iVH2 thiourea C=S NH2 I I NH2 I I NH2 urea c=o NH-CO I I alloxan CO CO 60 142 I I NH-CO COONa 116 I I COONa mesoxalate c=o -1 120 0.5 13 7 * reproduced by permission of the Biochemical Journal.7 reproduced by permission of the British J . Radiology WALTER M. DALE 297 function of the molecular groups constituting this molecule. The next six sub-stances however contain special groups of atoms which form a large part of the total molecule and these have a very marked and specific effect on the protective power. Such specific effects are masked by the average uniformity of the com-position of large molecules. Of special interest is the change of protective power caused by the substitution of a sulphur atom for an oxygen atom in urea and this has led to an investigation of other sulphur-containing compounds and of sulphur itself.14 Table 3 summarizes the results.Column 1 gives the weight of protector per ml used in the experiment and column 2 the respective sulphur content. Column 3 gives the protective power ( Q p ) per microgram of protector and column 4 the protective power Qs of such amounts of protector as contain 1 microgram of sulphur in each case. One can, therefore estimate from column 4 how the non-sulphur residue in any one com-pound affects the protective power of 1 microgram of sulphur contained in it, taking colloidal sulphur as reference. Elemental sulphur is about as protective TABLE 3 $ 1 2 3 4 QP QS thiourea 5.0 2.1 55 130 dimethylthiourea 5.0 1.54 18 58 sodium thiosulphate 5.3 1-06 (for one S) 24 118 p g protector pg sulphur per ml per ml colloidal sulphur 1.6 1-6 110 110 as thiourea and sodium thiosulphate but the introduction of two methyl groups into thiourea causes a considerable decrease in protective power of the sulphur The protective power of a protector can be expressed by the quotient, Q = (DP+I - Dz)/’DI - I/P, where Dr is the X-ray dose required to change the indicator by 63 % in absence of protector and Dp+ I the X-ray dose required to change the indicator by 63 % in presence of protector and I and P the concentration (pglrnl) of indicator and protector respectively.Q therefore represents the proportion of the X-ray dose taken up by the protector to that taken up by the indicator for equal weight of protector and indicator. In uncomplicated cases Q is independent of the concentration of protector used.In other cases however one finds that Q decreases with increasing concentration of protector as shown in fig. 1. Such a decrease of Q can be explained by assuming that energy taken up by the protector is handed on to the indicator. It is however not to be assumed that the decrease of Q is caused by a direct action of radiation on the protector molecule. Such a direct effect would require much higher concentrations of protector than those at which the decline of the protection effect becomes evident, and would lead to improbably high figures of the ionic yield when calculated on the basis of a direct mode of action. We have developed formulae based on the collision frequencies of a radical with protector and indicator molecules of given molecular weight which are valid for all cases regardless of the existence or non-existence of a handing-on effect and the values of the ratios of the probabilities of destruction of a radical by collision with a protector and an indicator molecule respectively are collected in table 4.The ratios pp/pR are a measure of the protective power per molecule of the protector for the given indicator. It will be seen that the protective power of thiourea is 10,000-fold greater than that of urea and further that a change from the indicator carboxypeptidase (C.P.) to the dinucleotide (D.N.) does not appreci-ably change the values of the protective power although these indicators are rather 1 reproduced by permission of the BritisJi J. Cnnccr. 298 PROTECTION EFFECT AND ITS SPECIFICITY different.It seems as if it were advantageous to use indicators which do not exhibit complicated concen trat ion-radia t ion dosage relationships. Finally the protection effect also operates in systems which are not true solu-tions. This has been shown in experiments with suspensions of biological units, TABLE 4 f protector mol. wt. p p l p ~ with C.P. p p l p ~ with D.N. thiourea sodium formate dime thy1 thiourea glucose dimethylurea egg albumin alloxan sodium mesoxalate sodium oxalate urea 76 68 104 180 88 -40,000 142 152 134 60 4-7 2.1 3-1 0.74 1.9 -0.79 0-21 0.4 -0.24 0.21 2.1 x 10-2 -2.4 x 10-2 -2.0 x 10-3 -7.5 :. 10-4 -e.g. bacteria and sperm of arbaciae to which various substances were added as protectors.Hollaender and his colleagues have used the survival rate of coli bacteria when irradiated in presence and in absence of alcohol SH compounds, etc.,ls and Evans and his colleagues 16 used the change of fertilizing power of arbacia sperm as indicator of radiation effects. Such experiments have clearly shown the protective action of substances dissolved in the surrounding medium. The interpretation however is complicated by the comparatively enormous 8 A Q 0 Curves are theoretical ; points expt. pg/ml ucose with C.P. 90 pg/ml \ 0 ‘a “ Changing quotient ” for C.P. A Jf dimethylurea with C.P. 30 B 0 glucose with C.P. 30 pg/ml Q conc. i n /ig,ml. \ size of the particles and the existence of cell membranes acting as permeability barriers and introducing surface phenomena.If the substance does not penetrate into the interior of the particle its effect is confined to competitive action in the surrounding fluid and the surface layers of the particle and can be understood by assuming that these surfaces play an active part in the maintenance of the bio-logical activities of the cell. A full explanation of the mechanism will therefore, require an analysis of permeability as well as an investigation of the question whether such surfaces can be the link between radiation and the effects observed. f reproduced by permission of the British J . Cancer WALTER M . D A L E 299 In contrast to systems of suspended particles is the condition of gels in which a colloid forms a continuous network of micelles between which the solvent circulates. Zt is important from the biological point of view that no protective action by the gelatinous network was observed by Skoog17 and Gordon and Quastler 18 when they irradiated Auxin in agar blocks or by Day and Stein 9 in their experiments with methylene blue in gelatine gels. These systems are of biological importance as models of the inhomogeneous physical structure of the interior of cells referred to in our experiments.13 1 Dainton and Holmes Nature 1950 165 266. 2 Evans and Urj Nature 1950 166 602. 3 Haissinsky and Lefort Compt. rend. 1950 230 1156; J . Chim. Phys. 1950 47 588. 4 Weiss Reunion Ann. SOC. Chim. Phys. (1951). 5 Fricke Cold Spring Harbor Symp. 1935 6 164. 6 Fricke Hart and Smith J . Chem. Physics 1938 6 229. 7 Fricke Symp. 4 (1950) Sept. 18-20 Army Medical Center Md.). 8 Fricke and Brownscornbe J . Amer. Chem. Soc. 1933 55 2358. 9 Stein and Day Nature 1950 166 146. 10 Forssberg Nature 1947 159 308. 11 Dale Brit. J. Rad. 1943 16 171. 12 Dale Biochem. J. 1942 36 80. 13 Dale Brit. J. Rad. 1947 Suppl. 1 46. 14 Dale Davies and Meredith Brit. J . Cancer 1949 3 31. 15 Burnett Stapleton Morse and Hollaender Proc. SOC. Expt. Biol. Med. 1951,77 636. 16 Evans Slaughter Little and Failla Radiol. 1942 39 663. 17 Skoog J. Cell. Cohp. PJiysiol. 1935 7 227. 18 Gordon and Quastler (private communication)