SIX FIGURES

Cytologists and geneticists work with raciiatioiis for two reasons. First, tlie cytogeiietic effect of radiations is an ex- tremely important factor in the total picture of radiation effects on organisms. Secoiid, it has seemed logical to hope that the study of radiation effects on tlic chroiiiosome-gene system would give important insight into thp 1iatur.c of that system. Radiation may be ayplied quantitatively nnd tlic cyto- genetic effects measured quantitatively. The hope is that by comparing tlie cytogeiietic effects with effects on simpler systems, the iiicchaiiisms of chromosomc cliangcs, including mutation, may be deduced. From these iiicchanisms sonietliiiig necessarily should be learned about the cytogmetic systems themselves. TJiitil recent years a paradoxical situation has cxisted wherein much more mas known about cytogeiietic ef- fects of radiations than about their effects on the supposedly simpler biochemical systems d i i ch should supply the a n s w rs to some of our problems. The paradox has centered arouiid the problem of radiation sensitivity. The biochemical systems that had been studied up until a few years ago could be ineas- urably affected only by radiation doses very much higher than those required to produce cytogeiietic effects. I t would there-

’ This work has been supported by a grant f rom the Coirimittc>e oil Groa tli, Na- tional Researell Coiinril, acting for tlic Anierirart C:inrer Society. Riucr tlie pres- eiitatiori of this paper , much of the material lies appeared in the Proceedings of the National Academy of Sciencev, 54 : 32d, 1948.

*Talk given by nr. X x ~ l a .

1 7 1

172 DANIEL MAZIA AND GERTEUDE BLUMENTHAL

fore seem unlikely that the primaq- actions of radiations on the chromosome-gene system were those on tlie previously studied biochemical systems. The paradox now appears to be only an apparent one. Thc work of Dale ( ’42, ’47), Barron ( ’48), arid others has now shown that many enzymes, even in solution, niay be affected hy sinall doses of ionizing radiations under special colditions ; where the enzyme is pui-e, and tlie solution is dilute aid free from other solutes. TTitli such sys- tems, a great deal is being learned about the primary actions of radiations on proteins, but m~lien one is speculating on radiation effects on chromosomes, these conditions are met with difficulty. Chromosomes are hardly dilute aqueous solu- tions of proteins. It secrns desirable to look for some other biochemical system whose organization is more closely com- parable to that in chromosomes and wliich is still sensitive to low radiation doses. The following discussion is not inteiided to discount the biological applicability of the work on dilute cnzymc solutions, but, 011 the contrary, to consider how, by varying the physical organization of the enzyme systcm, the mechanisms of inactivation of bi ocheniical systems at low doses may bc liberated from the limitations of a dilute pure aqueous solution.

The systcin described here became of interest as a conse- quencc of work on chromosome chemistry that has been going on iii our laboratory fo r a number of years. Observations on the action of enzymes on chromosomes led to the conclusion that thc fibrous physical organization, as well as the cheniical composition, of chromosomal proteins aff ectecl their digestion. This in turn led to a study of the relation between physical organization of an enzyme system and its activity. l’he case investigated was one which might exist in chromosoiiies, where the enzyme system is a “solid phase,” and where enzyme arid substrate are linked together in a solid organized structure. The particular case which seemed applicable to chromosomes was that of a surface film composed of eiizynie and substrate or a fiber prepared by compressing this film. T t developed in fact that the structural organization of such an e n s y e system

did affect its actirity. Our work on the characteristics of such enzyme systems has Inem piihlished (Rlazia ct al., '47) and will not he discussed here. I f the structural organization of such an enzyme system affects its activity, it might also be iiivolved in its radiation sensitivity. These experimerit s were aimed to test the possibility that structure was t? factor in radiation sensitivity, arid such being the case, to examine at a biochemical level some of the variables in chromosome sensitivity.

Fig. 1 Coiiiprcssed siirfaee film (fiber) of albunii~i, photogrn.phed through rrossed nicols. 9ppe;irmce of pepsin-albumin filiir is ideutical. Digestion is meas- ured as time of ilisnl~pearmice of these fibers.

Elwtron phot,omicrograpli of a portion of a compressed albulnin film, siiiiilar t o tlia t, shown in figure 1.

Fig. 2

EXPERTAIENTAL METHODR

A single experinlent is set up in the following ~vay. The starting material is a mixed solution of crystalline egg albumin and crvstalliiie pepsin (20-40 parts substrate to 1 part en- zyme). This ina terial is spread over an aqueous buffer of pH

174 DANIEL XAZIA AND GERTRUDE BLUMEhTTHL4L

4.0 in a surface-film tray. A monomolecular film composed of enzyme substrate is obtained. It is assumed that the enzyme- substrate ratio in the film is the same as in tlir original s o h - tion. The film is then irradiated with x-rays. To measure its activity, i t is compressed l o a fiber. Figure 1 shows the appear- ance of such a compressed pepsin-albumin film under the polarizing microscope; figure 2 a region of it under the elec- tron niicroscopc. I n figure 1 can be seen what appears to he a buiidle of birefringent fibrils, but figure 2 shows that the fiber is really a highly folded thin film. The experiment thus fa r has been carried out at pH 4, at ivhich the enzyme is inactive. The compressed film, or fiber, is now washed and transferred to HC1 at p H 1.8, the pH optimum of the enzyme. The fiber visibly digests itself a t this pH, and the time required for it to disappear is measured. This microscopic disappearance is really a proteolytic digestion rather than simple solution. This conclusion is supported by chemical measurements of digestion products of large amouiits of the compressed fiber made by Dr. Hayaslii and the author. This early worlr showed that the time required for disappearance of the fibers is a valid measure of the activity of the enzyme. The radiation work to be reported has heen done by the niicroscope method.

The film was irradiated with x-rays while still spread o n the surface. Digestion rate was measured after the film was compressed. The raw data of two series of x-ray experiments a re presented in figure 3. The curves represent rate of di- gestion as a fuiiction of dose for two types of film; one con- taining substrate and enzyme in a ratio of 40:1, and one in which the ratio is 20 :I. The term “coiiccntration” cannot Ine defined in such a solid system, therefore the relative amount of enzyme present is expressed hy these ratios.

Certain points are obvious from these IYLW data. First , effects are obtained with doses of 50-300 r, which are fairly low even for biological effects. Second, there appears to be a “threshold” for the radiation efiect, of which there is rela- tively little until a dose of about 50 r is reached. The effect is a slowing down of the digestion of the enzyme-substrate film.

INACTIVATION O F E N Z Y N E - S U B S T R A T E 175

6 -

5 - $

2 4 -

W

0 2

# W I- 3 g 3 -

2-

I -

The effect will be referred to as inactivation, not necessarily implying that the enzyme molecule itself is inactivated. While this is the most likely cxplanation, the whole enzyme sub- strate system is irradiated and it is conceivable that effects other than alteration of the enzyme molecule itself might slow down the digestion.

To an audience of radiation specialists, the dose-effect re- lationship in figure 1, where effect is measured by the digestion rate, is of limited interest. For purposes of comparison with

*

I I 1 I I 1 0 25 JO 75 100 125 150

DOSE - ROENTGEN U N J T S Fig. 3 Relation between x-ray dose in r uiiits and digestion rate of coinpressed

pepsin-albumin film. Ratios are albumin-pepsin ratios in solutions from which film were prepared.

176 DANIEL MAZIA AND GERTRUDE BLUXENTHAL

other data, and of theoretical treatment, the results expressed as percentage or number of pepsin units inactivated or sur- viving as a function of dose would be of greater value. The problem, then, is to translate experimental quantity - the rate of digestion - into numbers or fractions of innits affected by the radiation. This has been done by preparing calibration

I .o

.9

.8

g .7

6 - a

L

.6 W

I- 5: .5 I a .4

7 ..

.2

.I

40: I

\ 20: I -.-. -m

I I I I I I I

MINUTES TO DIGEST

I20

100

e

z 1c a z

60 5 m a m

80 $

W ..

-1

I

40

20

Fig. 4 Calibration curvcs for estinmting activity of pepsin-albumin 61111. Curve B rcprcsents enzyme substrate ratio in solutions from which film were prepared. Curves A, calculated from data of curve B, give relation brtween digestion time and enzyme content where the enzyme content of film having an albumin-pepsin ratio of 20 : 1 and 40 : 1 is taken as unity.

ITTACTITATTON O F ENZYME-SUBSTRATE 177

curvcs as shown in figure 4. Curve €3 relates the time of di- gestion, using the methods described above, to the ratio of albumin to pepsin molecules in the film. This is experinieiitally determined by measuring the digestion rate of film prepared from various substrate-enzyme mixtures. Once this data is secured the slowing down of digestion that results froni irra- diation can be expressed as though the radiation acted by rcnioving a certain number of active pepsin molecules from the system. Thus, if a 20: l film digests in 30 seconds before irradiation and in 3.5 minutes after irradiation, then, accord- ing to curve B, the after-irradiation digestion progresses at the same rate as a film containing 80 molecules of albumin to 1 of pepsin. Only 25% of the original activity remains. Curves A are calculated from the cspcrimental data of curve R, and give the relation between iiiactivation and digestioii time where substrate-enzyme ratios of 20 : 1 and 40 :1 are tal- Len as unity. It should be pointed out that the slowing down of di- gestion is treated as though the mechanism were a reduction in the number of active enzyme units. This cannot be statcd as fact, but tlic same criticism would apply to any experiment on enzyme inactivation where the amoiint of enzyme was esti- mated from activity measurements.

Figure 5 shows the same esperiincntal data as those in figure 1. Digestion times have been translated into surviving active fractions and plotted against dose. It is seen, in general, that the enzyme-substrate films are really sensitive to x-rays in absolute terms : dose of the 100 i' order hare effects equiva lenl to the inactivation of 50% of the active units originally present. It is seen that the threshold suggested in figure 1 is a real phenomcnon. The first increments of dose have rela- tively little cffect. Above about 50 r, the inactivation follows an exponential course. It is interesting that, even when the effects are plotted in terms of active fracfioms, entirely differ- ent curves arc obtained for the 20 :1 and 40 :1 film. This ob- servation is very difficult to explain on any of the current hypotheses of radiation action if the radiation has to affect the pepsin molecule itself. It would be easier to understalld if

178 DANIEL MAZIA b N D GERTRUDE BLUMElVTHBL

the sensitive unit is enzyme plus associated substrate, for then we would be dealing with sensitive units of different dimen- sions in the two cases. This observation is merely called to your attention. Considerably more experimentation will he required before it can be interpreted.

25 50 75 100 I25 I 50 DOSE- ROENTGEN UNITS

Fig. 5 enzyme. Data from figure 3 are in terms of the c:ilibration provided in figure 4.

Relation between x-ray dose in r units and surviving active fraction of

. The question of sensitivity should be considered a little

more fully. A very radiosensitive biochemical system is ob- served in these experiments, where doses that would be small for most biological effects a€fcct a large proportion of the units irradiated. A similar sensitivity could be demonstrated f o r a very dilute pure enzyme solution, but here it has been

INSCTIVATION O F ENZYME-SUBSTRATE 179

achieved in a system containing only enzyme molecules rela- tively close to each other in the abseiice of a protein sub- strate. The sensitivity in the present case seems to be a consequeiice of the molecular “topography” of the system. From the standpoint of the indirect action niechanisin of Dale and others, the prcseiit system would favor high sensitivity, for me have a masimuiii surface of exposure of the enzyme molcculcs to the irradiated medium, while the iionenzynie pro- tein all lies in the same plane as the enzyme and would no t be expected to act protectively. Bt the same time, the structural conditions introduced in the preparation of the system might affect its sensitivity. First, the protein molecules are at least partly unfolded in the film. Second, the molecules are linked to each olher in a continuous two-dimensional solid o r gel. Calculation of numbers of molecules inactivated really refer to mhat mere discrete molecules in the original solntion. It is possible that polymeric units of any size are being dealt with, and the high radiation sensitivity may reflect the possi- bility that the units being inactivated are large ones. Later some calculations bearing on this point will be given.

If the two-dimcnsioiial structural relations that were intro- duced on spreading the eiizymc-substrate system a t an inter- face arc responsible for the radiation sensitivity of the system, the seiisitivity might vary IT-; tli the physical properties of the film. The physical property that is most easily nianipulahle is the surface pressure o r the average area lper molecule. This is manipulated by sirnpl~7 compressing the film between two bar- riers. Figure G shows the eff’ect of cornpression on the inacti- ration produced by a giveii dose. The compression is expressed in units of area. Perhaps a more desirable expression would he in units of pressure; this is being done in most recent ex- periments which are not reportable a t this time. The curve shows the effects of 96 r as the film is compressed. A discoll- tinuorrs relatioilship between inactivation and area is seen. Up to ahout 10% compression, there is little effect. JVith further compression, the percentage inactivation by 96 r falls off rapidly, and at 28% compression effects of 7 7 r a re not de-

180 DANIEL ICTAZIA AND GERTRUDE BLUMENTHAZ

tectable. The sensitivity has been lowered but not destroyed : at 28% compression about the same inactivation is obtained with 240 r as with 96 r at 15% compression.

These experiments probably suggest that the high sensi- tivity of the enzyme-substrate film is somehow a consequence of the structural situation in the film. If the sensitivity of the system were dependent merely on its area of exposure to radiation products in the medium, there should be a simple

0 240r

PERCENT COMPRESSION Fig. 6 Relation between surfwe compression arid radiation effect. Compression

is expressed in units of area, not pressure. Curw represents inactivation by 96 r. Single point represents effect of 240 T at 28% compression.

INACTIVATION O F ENZYME-SUBSTRATE 181

proportionality between area and inactivation at a given dose. The complex relationship actually observed suggests a sensi- tivity factor that is imtrimsic in the system itself, and is vari- able by manipulation of the system. .Consideration of the possible intrinsic variables in radiation sensitivity is a matter of some biological interest in view of the fact that biological systems, such as chromosomes, do vary in radiation sensi- tivity under various internal and environmental conditions. Yet the treatment of radiation mechanisms has tended to be somewhat one-sided, to be more concerned with the physical probability of a primary radiation event in the system under study than with the conditions determining the consequences of the event, whether it be a “hit” o r tlie action of a radio- chemical product.

To the data of figure 6 may be added the observation that the compression effects are reversible. It is the reduction in rriolccular area or the increased pressure on the molecules that somehow governs their radiation sensitivity. Thc mecha- nism is a subject f o r speculation but the nest point to be con- sidered suggests that it may have to do with the area over which the effects of a primary physical event are spread.

Calculations of the extension in space of the consequences of a primary radiation event such as an ionization o r the pro- duction of a free radical are not new to this group. For in- stance, Dr. Kimball referred to a “sensitive volume” of micron dimensions, meaning that a priniary radiation event anywhere in such a volume would alter the whole unit in such a way as to be biologically detectable. The enzyme-substrate film permits us to consider in a simple case the possibility of the special spreading of a radiation effect. Following will be presented the results of some calculations based on the physi- cal constants in Lea’s hook, on a good many simplifying as- sumptions of our own, and on the data just presented.

First, the possibility that only ionizations in the film were effective have been considered; and these were calculated using Lea’s data for bulk ionization in protein, assuming the use of a 10-A! slice of a protein mass. The number of inactiva-

182 DANIEL MAZId AND GERTRUI)E RLUMENTHAL

tions was estimated from the data in figure 5 and the number of molecules in the film. The result was an order-of-magnitude discrepancy between the number of ionizations expected from a givcn dose and the number of pepsin units inactivated by that dose. Briefly, a yield of about lo3 pepsin units inactivated per ionization was obtained.

Second, consideration was given to the possibility that the inactivation of the film might be due to an indirect action mechanism such as has been considered evpcrimentally by Dale ('42, '47) and Barron ('48) and theoretically by Weiss ( '47). Only the case where some unstable radiation products such as free radicals which mediate the radiation effect is considered. Stable products such as peroxides can probably be excluded on the ground that previously irradiated medium is ineffective. The concentration of radicals that would be produced by a given dose has been calculated, and also the time required for a given number to diffuse to the surface, assuming in both cases that the radicals were stable. If every radical reaching the film were effective in causing an inactiva- tion, it would take something like 0.1 second for enough to reach the film. If only those radicals that would collide di- rectly with a pepsin molecule were considered, many seconds would be required. Rut, according to Lea, the half life of the radicals produced by irradiation of water is something like lop7 seconds. So, on either the direct o r indirect action theo- ries, the consequence of a single ionization would be the in- activation of a relatively large area of the film, one including many molecules. Whether one postulated an intermolecular spread would depend on his picture of the structure of the film. T t would be just as valid to propose that the unit struc- tures of the film are large polymeric units. On either view, the effects of a unit radiation event would be spread over a considerable distance. There are many biological results which would be explained by such a spreading effect. It is, after all, the case that the effects of radiation on chromosomes observed spread across the microscopic dimensions of chromosome threads. But, while niechanisms whereby radiation effects may

INACTIVdTION 0 1 7 EXZYME-SUBSTRATE 183

spread over microscopic distance are being sought, no great claims for these calculations are made, based as they are on so many simplifying assumptions and on constants that we are not in a position to judge critically.

No lengthy discussion lias been offered on the applicability of various mechanisms of radiation action because those vari- ables which are most significant in this connection have not yet been tested, particularly the dose rate and the relative effectiveness of various types of radiation.

STTMhI$RY

It has been shown that one may, using an enzyme-substrate film, set up a biochemical system which has a sufficiently high sensitivity to radiation to pwmii. comparisons with more coni- plex biological systems and which is adapted to the study of some of the variables, particularly structural variables, with 11-hich radiation biologists are concerned. The radiation sensi- tivity of the system depends on its structure and may be varied by physical manipulation so that some of the factors involved in intrinsic seiisilivity may be investigated. We have discussed the evidence that the high radiation sensitivity of this system may depend on an intermolecular spreading, over considerable distances, of the effects of a single primary radia- tion event. In the discussion to follow, some of the questions raised by these observations map be taken up more fully.

OPEN DISCUSSION

Questi0.n: I would like to know if, on the last slide, the com- pression makes any change in the time of digestion of the unrayed film.

MAZIA: I think this question is covered by our control ex- perimcnts. I t may he true, as the question suggests, that the previous history of the film determines its final dizestion rate even though the digestion rate is always measured after the film is conipletely compressed. TVe do find it nccessary to run control expcriments in which the procedure is exactly the same as in the radiation experiments but the film is not es-

184 DANIEL R I A Z I A AND GERTRITL)E ELUMENTHAL

posed to x-rays. Compression does not, in these control ex- periments, decrease the digestion rate.

FRIEDENWALD: May not the citrate buffer you are using be responsible for the effects, possibly through the forniation of organic peroxides?

MAZIA: I think not, unless the organic peroxides are un- stable, but more stable than radicals. As I mentioned earlier, there was no evidence f o r the action of stable radiation products. If anything, citrate should have lowered the sensi- tivity, since citrate niiglit conceivably exert a protective action.

Famo : As Dr. Jlazia pointed out, his results lead one natu- rally to think that the primary physico-chemical effect of the x-rays takes place in thc water and is then transmitted to the enzyme lying on the water surface. The transmission would presumably take place by diffusion of sonic active substance. On second thought, Dr. Xazia rejects this picture and is led to believe that the primary effect takes place in the protein film and is then transmitted by some unspecified mechanism. How- ever, this transmission through a solid material is not easily visualized. Hence it may be worth reviewing the validity of the argument which leads to rejection of the former picture. This we have done, Dr. Delbriick and I, briefly this morning arid x7e feel now that that argument should be discounted.

Dr. Mazia finds on p. 52 of the book on “Actions of radia- tions on living cells ” by D. E. Lea ( Maemillan, N.Y., 1947) that the half life of active water radicals produced by x-rays should be about 2 x lo-‘ seconds. During this short time such radi- cals could diffusc only over a distance of the order of 10 mu ; hence only active radicals produced within a water layer of such minute thickness could contribute efficiently to the enzyme inactivation. The number of activations produced within such a thin layer is inadequate to account for Dr. hIazia’s results. While the diffusion of activated water radicals is not the only conceivable channel of indirect action, no reason is seen at the moment f o r not taking it as a basis of discussion. I would rather fix my attention on Lea7s figure for the half life of active radicals.

I N ACTIVATION OF ENZYME-SUBSTRATE 185

In the first place, we see that the determination of the half life t through Lea’s equation TI-3, in which No,” is taken equal to 2, is affected to a very great extent by any er ror in the choice of the constant a in front of the logarithm, since the equation is solved by

t = (bZ/4D) (e erD/aTo - I).

Tn the second place the value used by Lea for the coefficient a

leads to a paradoxical result. According to Lea’s own theory of recombination of active radicals by diffusion ( s e ~ pp. 49-51 1.c.) the ratio a/8rrT) can be given a very simple interpretation. If an H and an OH radical start diffusing through an irifinite body of water from an initial distance r, they mould certainly recombine, provided r is no larger than a/8rrD. Now the value of a/SrrD obtained from Lea’s constants is Smp whereas it is clear that in practice there is always some chance of eseapiny reconibinati on, provided r exceeds the molecular dirncnsioiis. This means that the ratio cx/8rrD should not possihly exceed the molecular dimensions while it seems in fact to have been given by T,ea a value at least 30-40 times too large. TJsiny a reasonable value for a/8rrD in the preceding equation, the value of t is then found to be so extremely large that the whole picture of inactivation of radicals must be reconsidered. The initial objection to Mazia’s first picture seems thus to be re- moved.

I t may still he worth while to trace the origin of Lea’s choice of constants. It is seen from the rcfercnccs given in his book that this choice followed an early attempt to apply formulae from the theory of diffusion of molecules through gases to their diffusion through a liquid. This riietliod is seen to lead necessarily to a large error by excess in the estimate of a.

Qucs t io f i : To what extent would the enzyme in your system be denatured and how would this affect your calculations?

MAZIA: The term denatnration has a certain ambiguity in this case. Spreading a protein at an interface involves a drastic rearrangement of the molecule and loss of solubility, hence the term “surface denaturation. ” Yet surface chemists

186 DANIEL MAZIA AND GERTRUDE BLUMENTHAL

feel free to discuss the question of whether a surface-dena- tured protein has lost its activity. In our system, we think we have a case of biological specificity surviving surface de- naturation. Insulin is another case. I did not intend to imply that we 11-ere sure that all the pepsin spread was in the native state originally or remained so on spreading. Our figures give R niaxiiiium, based on the weights of the original crystalline pepsin preparation. As to the effect of possible denatnr a t' ion of some of the pepsin in our calculations, the actual figures would be affected but not the conclusion that the radiation effect was spread over a considerable area. The calculations show a discrepancy between the number of ionizations any- where i~ the fiZm or the number of radicals impinging alzy- where 02% the film and the number of inactivations. I f the total number of active pepsin molecules were smaller than calcu- lated, they would be proportionately farther apart, so that even though the number of activations \vould be less, the area covered by a given number of unit radiation events would be the same. If we were in position to calculate the number of direct hits on active pepsin molecules, whether direct ioniza- tions o r through radicals, the chances of a hit would decline with the number of targets, a i d the discrepancy would be the same o r worse.

LITERATURE CITED

DALE, W. 31. 1947 Action of r aha t ion on aqueous solutions: Experimental work with enzymes in solution. Brit. J. Radiol. Suppl. 110. 1: 46.

The cffeot of X rays on the conjugated protein d-amino-acid oxidase. Bioehem. J., 36: 80.

BAILEON, E. S. G. 1948 The effect of ionizing radiations on the actibity of cn- z p e s . Rrookhaven Conference Report BXL-C-1. p. 10.

MazlA, D., T. HAIASHI AND K. L. YUDOWITCH 1947 Fiber structure in chromo- somes. Cold Spring Harbor Symp. Quant. Biol., 12: 122.

WEISS, J. 1947 Some aspects of the action of radiations 011 aqueous solutions. Brit. J. Radiol. Suppl. no. 1 : 56.

1942