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The inactivation of catalase by deuterons and heat
The Inactivation of Catalase by Deuterons and Heat R. B. Setlow From the Biophysics
Division,
Yule University,1
Received
New Haven, Connecticut
June 11, 19.51
Data on the inactivation of molecules of biological importance by radiation may yield two types of information: theories of the action of radiation on large molecules, the “target theory” of ionizing radiation action (I), on the one hand, and the structural make-up of large entities such as viruses, on the other (2). One way to simplify the difficulties in interpretation of the results of ionizing radiation action is to irradiate dry aggregates of particles. Such a technique greatly reduces, if not completely eliminates, Ohe indirect effects of radiation associated with t,he production of activated radicals or toxic products in water. We shall concentrate on the direct’ action of radiation. The interpretation of irradiation results is further simplified if one uses highly ionizing fast particles. In this case, the ionization is confined to a narrolv region surrounding the path of the particle. For these reasons we have used deuterons to bombard samples of dried catalase. The choice of cat>alasc as the substance to be irradiated was dictated by the ease of determining its activity and the knowledge that it was a molecule large enough so that any gross internal structure might be apparent under deuteron bombardment. As a starting point, it is assumed that there is a close correlation between enzymat,ic activity and molecular st’ructure. This seems justified in the case of the hemoproteins (3). In an effort, to correlate the mechanism of ionization and heat inuctivation of catalase, data were obtained on the rate of inactivation of dry catalasc as a function of temperature.
The catalase used in these investigations was purchased,2 and according to t hct supplier was derived from I)eef red cells. Activit,y was tl&rmined by mrasuring the* ---__-___--~ ~~~~-~ ~--1 -4ssisted in part by the U. A. At,omic Energy Commission. 2 .4rmour Powdered Catalase :30, from Armour and Co., Chicago, 111. 396
ISSCTIVATION
OF
C.1T.kL.kSE
397
amount of Hz02 decomposed in a standard time (4 min.) and then comparing this figure with those obtained from a previously constructed calibration curve. The following procedure is essentially that, given by the supplier. A cntalxse sample for assay was mixed with 25 ml. of 0.209J H,O?, which was also a phosphate-c:itlntf, buffer at pH 7.0. The mixture was kept :lt, O’Y’. for 4 min., after \vhich the reacbtion was st,opped wit,h 2.5 ml. of 10% H,SO,. The amount of H302 remaining FY:LStl~trrmined by adding an excess of ICI and titrating the resulting 12 with 0. I N Sn&Oa. The amount of Hz02 decomposed was then found 11).c~omparison with a t)l:tnk run in which the acid was added first. The technique of cyclotron bombardment of dry preparations has been described elsewhere (2). The big difficulty in this work is in ensuring that the deutcron IuL~~ from the cyclotron is uniform in cross section. The mean energy of the tleuterons from the cyclotron was 3.76 m.e.v. This energy could be decreased, and the distjance-rate of energy loss increased, by interposing thin aluminum foils in front of the samples. The deuteron current was measured on a galvanometer, ad t,11e:troa of the bc:m was determined from the blackened region on a piece of photographic paper put into the beam. These figures plus the known time of irradiation give the number of dcuterons/cm.z st.riking the sample. Since t,he samples for homt~nrdmcnt ‘scrc thin (-0.1 mg./cm.2), the change in dcuteron energy in traversing the sample ~vas small compared to its total energy, and only a very small correction is ncedcd in computing the energy loss. The figures for the energy loss of deuterons of the different energies used in this work have been obtained from an article by Livingston and Bethe (1). The samples for deutcron bombardment were prepared by spreading 0.05 ml. of a cat,alase n-ater solution, containing 0.10 mg. of solids, over glass cover slips of 1 cm. diameter. The samples were dried in a vacuum desiccator. They tverc t hc:n placrti in the vacuum (p < 10m4mm. Hg) bombardment chamber of the cyclol.ron and irradiated. Irradiation times were from 5 sec. to 3 min. at beam currents of about 4 X 1O-8 amp. Control samples were t,reated similarly but were not irradiated. There s-as negligible loss of activity on drying. After irradiation t,he sample cover slips were placed in 10 ml. of quartz-distilled water, and the dried catalasc was allowed fo dissolve for 5 min. One ml. of this solution was assayed for catalytic :tc:tivit.y as outlined above. The samples for the heat-inactivation studies were prepared and assayed as \VVR: those for cyclotron irradiation, except for the following differenres. For the runs at temperatures above lOO”C., the samples were dried on aluminum foil to ensure rapid heating. The samples were dried for about an hour in a vacuum desiccator to which :t liquid air or Dry Ice trap was connected. If the samples were not dried in this fashion, the results obtained were not internally consistent. The oven temperature was kept constant to within A 1°C. Opening the oven ~OOI lo remove samples resulted in a large temperature drop which was somewhat mitig:tt,cd by using heavy metal plates under t hc COWI slips.
Oeuteron Ivadiation
Results
The redts of bombarding dry catalase by deuterons of 3.76 m.e.v. energy are shown in Fig. I ! where the per cent remaining activity on a
398
I<. 13. SETLOH
logarithmic scale is plotted against the number of deuterons/cm.” striking the sample. The resulting curve is seen to be a straight line, which means that if we let Ao be activity of the controls and A the activity after delivery of a dose of D deuterons/cm.2, then In $- = -SD, or 0
A - = &3”. Ao
The quantity S which appears in these equations is expressed in cm.2/ deuteron, and will be spoken of as the inactivation cross section of the molecule. These equations will not be derived here, but will be recog100
50
20 5 i= Y
IO
';' i= s s 5
2
I
I 3
I I I I I 6 9 12 15 16 DEUTERONS PER SQUARE CENTIMETER
2lxW
E’ro. 1. The relative sctivit,y of catalase on a logarithmic scale vs. the number incident drut.erons/cm.*
of
nized as describing a single-hit inncativution process. The inactivation cross section corresponds to the probability that the passngc of a single deuteron through the molerule will inactivate it. .2n inactivation cross section eqllal t,o the geomctriwl (*ross sect ion (the average projwtctl arca), as determined by such methods as difl’usion and sedimentation? means t,hat t,he entire molecule is sensitive to the passage of a deuteron through it. For a relative acti\rity of V’ (3i('(), the above equation gives ,SD = 1. The inact,ivation cross section is thus the reciprocal of t’he dose for 37yC relative acti\-ity, and from Fig. I S = (7.1 X 1013deuterons!/c~m.S)~-l = I .Jl X IO--‘” cm.” = l,-klO A.?. The molecular weight of catalasc from sedimentation-dift’usion experiments is thought to he in the neighborhood of 250,000 (3), with an axial ratio, on an ellipsoidal model, near 3 to 1. From these data and using a density of 1.33 g./cm.“, one may calculate the size of the ellipsoid as 174 A. by 58 A. The projected area of such an ellipsoid, averaged over all orient,ations, is easily shown to be about GO00A.?. Electron microscopy of crystalline liver catalase gives a similar molecular weight and dimensions, on a parallelepipcd model, of Gi X 64 X 80 A. (6). While these dimensions of the two models differ, bot’h give about the same average projected area. From the large difference between 6000 A.3 and 1410 A.2 it is concluded that the passage of a deut,eron through a catalasc molecule will not completely inactivak it. Howcwr, there is a good probability that, a deuteron may pass through a cat&w molecule without producing any physical effect whatsoever. .4 fast, charged particle may lose energy in three ways: eit,her by ionization, escit,ation, or nuclear recoils. The lat’ter accounts for a negligible frwtion of the energy loss at high partiolc vclocGt,ies. The loss of energy by cscitat’ion and ionization has recently been considered by Pollard and Forro (i), who have shown that the energy associat,ed with a primary ionization is 9.5 e.v., and that this is accompanied by an average of two cscitations of 10 e.v. each. Since pract,ically nothing is known of the oU’ect of a IO-e.v. excit’ation on proteins, x-e will not, consider t’hcm, even though they may represent an important, part of t’he inactivation. At 6he deuteron energies we are considering, 3.76 m.e.v., the average energy loss is 233 e.v./lOO A. of protein. This corresponds to about two primary ionizations/100 A. Since the average depth of a catalase molecule whose dimensions are given above is about 50 A., a deuteron striking the molecule mill produce an average of one primary ionization
4-00
II. H. SETLOW
0’ Dlffuslon - Sedimentation
crwf
section: 6~ Id A’
.o -
O-
O-
O-
0 0
I 100
I I I I 200 300 400 500 Energy Loss in ev per 100 i of protein
1 600
wit#hin the molecule. The linear distribution of these primary ionizat)ions is random. Hence if we call z the average number of ionizations pel molecule, then the probabilit,y of n ionizations per molecule is given t)y the Poisson expression : P(n)
Zne-s = ___ n. I .
The chance of no ionizat’ion in the molecule is Ed, which for our case is 359& Clearly the chance of a deuteron not producing a primary ioni-
zation is not large enough to explain why t,he innctivat’ion cross sect ion is four Gmes smaller than the geomet,ricnl cross section. In view of the wide discrepancy it was advisable to obt.ain inact,iration data at scvernl higher ionization den&es. This was done by decreasing the deutcron energy by means of aluminum foil and determining the per cent act,i\-it) after bombardment. The results of these runs are shown in E’ig. 2, where the inactivation cross section is plotted against, t’he rate of energ! loss. The solid line is a theoretical curve calculatccl, by use of the Poisson expression, for a spherical target whose cross section is about 3400 8.’ [cf. Ref. (l), p. 3531. The data do not fit t.his simple theor) well, but there is no doubt that the inactivation cross sect’ion is less than the size of a molecule of 250,000 molecular weight. A1t 530 e.v.!’ 100 A. the chance of no primary ionization in such a molecule is about, SC;, while the chance of one hit, is 13%>and of more than one hit SO?;,. Resdfs of Tempcratrrre Inactivation The inactivation of dry catalase by heat follows first-order reaction kinetics. The logarit,hm of the relative activit.y is proportional to the time. Typical
data are shown in Fig. 3. Thus $
= e+t,
where k, is
0
the specific rate constant. Dry heat inactivat,ion does not involve collisions between catalase and other neighboring molecules, but merely the increased vibration of intramolecular bonds. Thus it seems reasonable, t,hough not necessarily certain, that the purity of the preparation should have no effect on the inactivation rat’e. Dry heat inactivation is seen to require much higher temperatures than is visually required for solutions of enzymes. It should be noted that some of the curves of Fig. 3 do not pass through the 100% point. This is the result of insufficient drying of the samples. Initially these semiwet samples inactivate at a very rapid rate, but after some of the mater is driven off they follow the much slower inactivation rate typical of dried material. The theory of absolute reaction rates (8) gives for the specific rate co&ant kl:
where K is Boltzmann’s constant; Ir, Planck’s constant; T, the absolute temperature; R, the gas constant/mole; and AFt is the free energy
402
Il.
B.
SETI,O\J~
0 l
95 5-c. 85.5-c.
A 78 5'c. n 116 0-C
20
60
40 TIME
I
I
I
a0
100
120
IN MINUTES
of act,ivation for inactivat,ion. Thermodynamics gives AF:, in t.rrms of the heat of activntjion LIH$ and the enkopy of wtiv:Ltion A#, as AI;T z AH: - 7’ASf
(2)
11 graph of Apt, c~nlculated from the data of Fig. 3 and ICq. (I), versus absolute temperature is shown in Fig. 1. The error in these detjerminations is principally t,he tcmperaturc fluctuations in the oven, since :I 1°C. error in the o\-cn temperature is abollt, a, 0.35; (Arror in the calrulatrd valtlc of AFT. Errors in I;, are negligilk since t,his term itppears
27.600
2 27.400 \E
27.200
27.000
26.600
f?JG.
I
I
I
340
350
360
370
Temperature
'K
1. The free cnergg of actiw~tion,
,
4I ‘1, of dr\- atalnse ternperat.uw.
360
plottd
390
against the absolute
404
R.
B.
SETLOW
The values of A@ and AS obtained from Fig. 4 and Eq. (2) arc shown in Table I. For comparison there are shown similar values for heat inactivation of catalase in wat,er. Since the enzyme preparation was not pure, the latter values have dubious significance. They arc included to emphasize tjhc difference Mween the kinetics of wet and dry inactivation. Reszdts of Combined Ileuteron and Neaf Inactivation Some of t,he deuteron data, taken after the results presented in Fig. 1 were obtained, gave inactivation cross sections somewhat higher t’han
1
DEUTERONS FIG. 5. The relative
PER SQUARE CENTIMETER
activity of part~ially heat-innct,iwted rat:tlase on a lognrithmic scale VS. the number of inrident dcuterons/cm.?. 0 - 0 -, experimental points; - . - . -, resolution into t,n-o straight lines; ~~ . ~~ , curve for unheated catalnse (Fig. 1).
the 1410 A.” previously found. It was suspected that this was the resuk of partial heat inactivation of stock ratalnsc. This supposition was verified by bombarding catalase with deuterons after preliminary dry heat, inactivation (at 91%. for 30 min.) and by heat inactivation of bombarded samples. The results of these experiments were the same. The curve of per cent, activity versus deut8eron dose show~l a high init’ial slope much larger than for deut,erons alone. The data are shown in Fig. 5. The shape of the curve drawn through the experimental points ~-as the same whether the cat’alasr was heated before or after bombardment. The inactivation curve may be resolved into the sum of two straight lines (which on a linear plot is the sum of two exponenGals). The cross sections corresponding to t,hese components arc 8700 *4.” and 1410 A.‘. The latter value is found with deuteron bombardment alone. The former is greater than the geometrical cross section. If we take a literal view of the component curves of Fig. 4, \ve can say that 00$$0 heat inact’ivation yields three classes of catalase. The altered but still first is unchanged (l’i70), the second is structurally retains its cat,alyt,ic activit’y (237;) and the t,hird is completely inacbirated (607,). Drscx~ssrox
AND
I~TERPIWTITION
The dat,a for the heat inactivation of dry catalase have a simple qualitative interpretation. An increase of ent’ropy of a system is associated with a more random configuration. The denat’uration of prot,eins in solution shows an increase in entropy for the act’ivated complex (9). This more random configuration has been associat’ed with a partial unfolding of the protein molecules. From this partially unfolded activated &ate the molecule proceeds to a still more unfolded final configuration. In our experiments t’here was no evidence of a reversible reaction taking place, so that we can only infer characteristics of t’he activated state but not the final state. The decrease in entropy between the initial and activated states of dry catalase is a definite indication that the molecule is not unfolded. Beyond this, the physical significance of I he negative entropy change is not clear. One more point may be made about the process of dry heat, inactivation. The transition from ordinary stable dry cat’alase to the a&i\-ated state is not a one-step process. There exists at least one intermediate configuration which has a high relative stability. This intermediate state shows up with an abnormally high deuteron inactivation cross
-406
II. B. SETLOIV
section. It) is relevant that, three types of catulase ha\-c Iwen found by spectroscopic analysis ( 10). While it is tempting to speculate further concerning the prowss of heat, inactivat,ion, the present data do not, seem to warrant it. Two possible inberpretations of the deuteron inactivation data will be given. The first. explanation is based on t,he assumption that the molecular Iveight of catalase is about 250,000. If this is true, then an inactivation cross se&ion less t,hnn the molecule’s projected arca may mean that’ t*he molecule can only be inactivated if the energy rcleascd by a deuteron appears in certain critical regions of the molecule, or that the passage of a deuteron knocks out only half the catalyt,ic :wtivity of the molecule. A%tJ t’he higher ionization densit,ics, several primary ionizations are produced within the molecule. I~ccausc of the large mass of the deuteron compared t,o the electron clouds through which it passes, the deuteron travels along a st)raight line and the ionizutJions it produces are along this path. One might guess t’hat a deuleron must liberate energy close to one of the four hemc groups if the catalytic activity is to be destroyed. i.e., catalytic activity is not, a gootl intlicatol of catalase structure. In t)his view, a partially heat-inactivat*ccl molwl~lc would have several broken bonds distributed at random, and while still stable against inactivation by thermal energy (about l/30 e.v.), the release of about, 100 e.v. by a tlcuteron would br enough to (wry t,ho molecule to the nctivat,ed siatc anal on to an innc:t.ive configuration. The deut,cron woss section for the partially heat-inac:t.i\.at,ctl wmponent’ is larger than the molecular size. h deuteron passing ocs~sitlc t’he molecule can inactivate it,. The discrepancy may be the result’ of two factors. The events resulting from a passing deut,eron art’ not strictly confined to a lint bllt, are tlistributlerl \vithin about 3 :2. at~rmtl the deuteron’s pnt.h (7). This is simply ;L rtsult of t,hc fac:(, 1h:it, the! electric field of a charged patticlc does not, ubrupt,ly fall to zero for distances which are large compared to the particle size. If \w add 3 ;2. to all the dimensions of a cat,:tlase molecule, then t,hc ctiwtive wwu within which a deutcron may cause inact’ivation is about 7000 A.“. The steep inactivat,ion curve is obt.xincd by taking t.he diffcrcnw bct~wccn two curves (Fig. 5). The error thercforc, is larger than for 4thcr of t hc other two and may be at, least f 20’:{,. There is one important object,ion to the explanation given above. The effects of heat plus deuteron bombardment. were found for 3.70 m.e.v. deuterons. At, this energy, as remarked before, there is a 35’;/,
chance of a deuteron producing no primary ionization within a catalase molecule. Unless the effectiveness of excitations and secondary electrons is large, we would expect always to obtain an inactivation cross scction smaller than the geometrical one. We are led to a second possible int’erpretation, based on the assumpt’ion that catalytic activity is a good indicator of catalase structure. If this is true the molecular weight’ of catalase is considerably less than 250,000. If we take 3400 A. for the cross section of the molecule (see Fig. 2), then the molecular weight for the equivalent sphere is 130,000. It should be noted, however, that the theoretical curve and the experiment,al points of Fig. 2 are not in good agreement. While the heatinactivation data still give a picture of intermediate configurations preceding the activated state, the combined deuteron and heat effect can no longer be explained in terms of raising the particle of 130,000 molecular weight to the activated stat)e, since such a particle cannot have a 8800 A4.3cross section unless it is very asymmetrical (an axial ratio of about 50 to 1). This large cross section may be the result of indirect action. Under the action of heat, a catalase molecule may be in an intermediate configuration. It still has catalytic activity, but is more sensit’ive to radiation. The effect of an ionizubion within the molecular confines may now cause addiCon changes in intra,molecular forces, so that’ subsequent dissolving in water allows the molecule t,o unfold. If the molecule unfolds, oxidizing groups may now be available to act upon intact catalase molecules. Alternatively the inactivated molecule may serve as a nucleus for aggregation of active molecules. This would represent a decrease in cstalase solubility. Evidence for a decrease in soluhility of hemoglobin after deutcron bombardment has been found by Appleyard (II). In either of these cases a deuteron can miss a molecule and still inact>ivate it, and a cross section larger than that of a single molecule would be observed. It is of interest that a bacterial virus shows a somewhat similar behavior toward a combination of dry heat and deuteron irradiation 02). It is hoped that the data and discussion presented here illustrate the use of physical inactivation methods in investigation of the st,ruct,ure of large molecules. AC~NO~~LEDGME~YTS
The author wishes to thank FT. R. Adams, for his help in running the cyclotjron, nnd the other members of the Biophysics Division, for many helpful discussions.
SUMMARY
The inactivation of beef red cell cat?lase by fast dcuterons, heat, and a combination of the t,wo has been reported. The data have been analyzed quantitatively in terms of the target theory of ionizing radiation and the theory of absolute reaction rates. Evidence for the existence of at least two forms of stable catalase has been presented. The data are shown to indicate two possible alternative molecular weights (about 250,000 and 130,000) for catalase. REFERENCES
1.
LEA,
2. 3. 4. 5. 6. 7. 8.
POLLARD, E. C., Am. Scientist 39, 99 (1951). GRANICK, S., AND GILDER, H., Advances in Enzymol. 7, 305 (1937). LIVINOSTON, s., AND BETHE, H. A., Rev. Modern Phys. 9, 263 (1937). SUMNER, J. B., AA-D GRALEN, N., J. Biol. Chem. 125, :3X (1938). HALL, C. E., J. Riol. Chem. 185, 749 (1950). POLLARD, E. C., AND FORRO, F., Arch. Biochem. Hiophys. 32, 256 (1951). GLASSTONE, S., LAIDLER, K. J., AND EYRIKX, H., The Theory of R:tt)e Prowsses.
1). E., Actions of Radiations on Living Cells. .\lux~illari,
McGraw-Hill,
New York, 1947.
New York, 1941.
0. STE~RN, E. k, Advances in, Enzymok 9, 25 (1949). 10. BROWN, G. L., cited by Randall, J. T., Proc. Ro?/. Sac. (London) 11. AWLEY~RD, It. Ii., Phys. Rev. 83, 231 (1951). 12. ADAMS, w. I<., AND POLLARD, P:., Ph.+% II%v. 83, 2:SO (i!);il).
A208, 1 (1951).
Division,
Yule University,1
Received
New Haven, Connecticut
June 11, 19.51
Data on the inactivation of molecules of biological importance by radiation may yield two types of information: theories of the action of radiation on large molecules, the “target theory” of ionizing radiation action (I), on the one hand, and the structural make-up of large entities such as viruses, on the other (2). One way to simplify the difficulties in interpretation of the results of ionizing radiation action is to irradiate dry aggregates of particles. Such a technique greatly reduces, if not completely eliminates, Ohe indirect effects of radiation associated with t,he production of activated radicals or toxic products in water. We shall concentrate on the direct’ action of radiation. The interpretation of irradiation results is further simplified if one uses highly ionizing fast particles. In this case, the ionization is confined to a narrolv region surrounding the path of the particle. For these reasons we have used deuterons to bombard samples of dried catalase. The choice of cat>alasc as the substance to be irradiated was dictated by the ease of determining its activity and the knowledge that it was a molecule large enough so that any gross internal structure might be apparent under deuteron bombardment. As a starting point, it is assumed that there is a close correlation between enzymat,ic activity and molecular st’ructure. This seems justified in the case of the hemoproteins (3). In an effort, to correlate the mechanism of ionization and heat inuctivation of catalase, data were obtained on the rate of inactivation of dry catalasc as a function of temperature.
The catalase used in these investigations was purchased,2 and according to t hct supplier was derived from I)eef red cells. Activit,y was tl&rmined by mrasuring the* ---__-___--~ ~~~~-~ ~--1 -4ssisted in part by the U. A. At,omic Energy Commission. 2 .4rmour Powdered Catalase :30, from Armour and Co., Chicago, 111. 396
ISSCTIVATION
OF
C.1T.kL.kSE
397
amount of Hz02 decomposed in a standard time (4 min.) and then comparing this figure with those obtained from a previously constructed calibration curve. The following procedure is essentially that, given by the supplier. A cntalxse sample for assay was mixed with 25 ml. of 0.209J H,O?, which was also a phosphate-c:itlntf, buffer at pH 7.0. The mixture was kept :lt, O’Y’. for 4 min., after \vhich the reacbtion was st,opped wit,h 2.5 ml. of 10% H,SO,. The amount of H302 remaining FY:LStl~trrmined by adding an excess of ICI and titrating the resulting 12 with 0. I N Sn&Oa. The amount of Hz02 decomposed was then found 11).c~omparison with a t)l:tnk run in which the acid was added first. The technique of cyclotron bombardment of dry preparations has been described elsewhere (2). The big difficulty in this work is in ensuring that the deutcron IuL~~ from the cyclotron is uniform in cross section. The mean energy of the tleuterons from the cyclotron was 3.76 m.e.v. This energy could be decreased, and the distjance-rate of energy loss increased, by interposing thin aluminum foils in front of the samples. The deuteron current was measured on a galvanometer, ad t,11e:troa of the bc:m was determined from the blackened region on a piece of photographic paper put into the beam. These figures plus the known time of irradiation give the number of dcuterons/cm.z st.riking the sample. Since t,he samples for homt~nrdmcnt ‘scrc thin (-0.1 mg./cm.2), the change in dcuteron energy in traversing the sample ~vas small compared to its total energy, and only a very small correction is ncedcd in computing the energy loss. The figures for the energy loss of deuterons of the different energies used in this work have been obtained from an article by Livingston and Bethe (1). The samples for deutcron bombardment were prepared by spreading 0.05 ml. of a cat,alase n-ater solution, containing 0.10 mg. of solids, over glass cover slips of 1 cm. diameter. The samples were dried in a vacuum desiccator. They tverc t hc:n placrti in the vacuum (p < 10m4mm. Hg) bombardment chamber of the cyclol.ron and irradiated. Irradiation times were from 5 sec. to 3 min. at beam currents of about 4 X 1O-8 amp. Control samples were t,reated similarly but were not irradiated. There s-as negligible loss of activity on drying. After irradiation t,he sample cover slips were placed in 10 ml. of quartz-distilled water, and the dried catalasc was allowed fo dissolve for 5 min. One ml. of this solution was assayed for catalytic :tc:tivit.y as outlined above. The samples for the heat-inactivation studies were prepared and assayed as \VVR: those for cyclotron irradiation, except for the following differenres. For the runs at temperatures above lOO”C., the samples were dried on aluminum foil to ensure rapid heating. The samples were dried for about an hour in a vacuum desiccator to which :t liquid air or Dry Ice trap was connected. If the samples were not dried in this fashion, the results obtained were not internally consistent. The oven temperature was kept constant to within A 1°C. Opening the oven ~OOI lo remove samples resulted in a large temperature drop which was somewhat mitig:tt,cd by using heavy metal plates under t hc COWI slips.
Oeuteron Ivadiation
Results
The redts of bombarding dry catalase by deuterons of 3.76 m.e.v. energy are shown in Fig. I ! where the per cent remaining activity on a
398
I<. 13. SETLOH
logarithmic scale is plotted against the number of deuterons/cm.” striking the sample. The resulting curve is seen to be a straight line, which means that if we let Ao be activity of the controls and A the activity after delivery of a dose of D deuterons/cm.2, then In $- = -SD, or 0
A - = &3”. Ao
The quantity S which appears in these equations is expressed in cm.2/ deuteron, and will be spoken of as the inactivation cross section of the molecule. These equations will not be derived here, but will be recog100
50
20 5 i= Y
IO
';' i= s s 5
2
I
I 3
I I I I I 6 9 12 15 16 DEUTERONS PER SQUARE CENTIMETER
2lxW
E’ro. 1. The relative sctivit,y of catalase on a logarithmic scale vs. the number incident drut.erons/cm.*
of
nized as describing a single-hit inncativution process. The inactivation cross section corresponds to the probability that the passngc of a single deuteron through the molerule will inactivate it. .2n inactivation cross section eqllal t,o the geomctriwl (*ross sect ion (the average projwtctl arca), as determined by such methods as difl’usion and sedimentation? means t,hat t,he entire molecule is sensitive to the passage of a deuteron through it. For a relative acti\rity of V’ (3i('(), the above equation gives ,SD = 1. The inact,ivation cross section is thus the reciprocal of t’he dose for 37yC relative acti\-ity, and from Fig. I S = (7.1 X 1013deuterons!/c~m.S)~-l = I .Jl X IO--‘” cm.” = l,-klO A.?. The molecular weight of catalasc from sedimentation-dift’usion experiments is thought to he in the neighborhood of 250,000 (3), with an axial ratio, on an ellipsoidal model, near 3 to 1. From these data and using a density of 1.33 g./cm.“, one may calculate the size of the ellipsoid as 174 A. by 58 A. The projected area of such an ellipsoid, averaged over all orient,ations, is easily shown to be about GO00A.?. Electron microscopy of crystalline liver catalase gives a similar molecular weight and dimensions, on a parallelepipcd model, of Gi X 64 X 80 A. (6). While these dimensions of the two models differ, bot’h give about the same average projected area. From the large difference between 6000 A.3 and 1410 A.2 it is concluded that the passage of a deut,eron through a catalasc molecule will not completely inactivak it. Howcwr, there is a good probability that, a deuteron may pass through a cat&w molecule without producing any physical effect whatsoever. .4 fast, charged particle may lose energy in three ways: eit,her by ionization, escit,ation, or nuclear recoils. The lat’ter accounts for a negligible frwtion of the energy loss at high partiolc vclocGt,ies. The loss of energy by cscitat’ion and ionization has recently been considered by Pollard and Forro (i), who have shown that the energy associat,ed with a primary ionization is 9.5 e.v., and that this is accompanied by an average of two cscitations of 10 e.v. each. Since pract,ically nothing is known of the oU’ect of a IO-e.v. excit’ation on proteins, x-e will not, consider t’hcm, even though they may represent an important, part of t’he inactivation. At 6he deuteron energies we are considering, 3.76 m.e.v., the average energy loss is 233 e.v./lOO A. of protein. This corresponds to about two primary ionizations/100 A. Since the average depth of a catalase molecule whose dimensions are given above is about 50 A., a deuteron striking the molecule mill produce an average of one primary ionization
4-00
II. H. SETLOW
0’ Dlffuslon - Sedimentation
crwf
section: 6~ Id A’
.o -
O-
O-
O-
0 0
I 100
I I I I 200 300 400 500 Energy Loss in ev per 100 i of protein
1 600
wit#hin the molecule. The linear distribution of these primary ionizat)ions is random. Hence if we call z the average number of ionizations pel molecule, then the probabilit,y of n ionizations per molecule is given t)y the Poisson expression : P(n)
Zne-s = ___ n. I .
The chance of no ionizat’ion in the molecule is Ed, which for our case is 359& Clearly the chance of a deuteron not producing a primary ioni-
zation is not large enough to explain why t,he innctivat’ion cross sect ion is four Gmes smaller than the geomet,ricnl cross section. In view of the wide discrepancy it was advisable to obt.ain inact,iration data at scvernl higher ionization den&es. This was done by decreasing the deutcron energy by means of aluminum foil and determining the per cent act,i\-it) after bombardment. The results of these runs are shown in E’ig. 2, where the inactivation cross section is plotted against, t’he rate of energ! loss. The solid line is a theoretical curve calculatccl, by use of the Poisson expression, for a spherical target whose cross section is about 3400 8.’ [cf. Ref. (l), p. 3531. The data do not fit t.his simple theor) well, but there is no doubt that the inactivation cross sect’ion is less than the size of a molecule of 250,000 molecular weight. A1t 530 e.v.!’ 100 A. the chance of no primary ionization in such a molecule is about, SC;, while the chance of one hit, is 13%>and of more than one hit SO?;,. Resdfs of Tempcratrrre Inactivation The inactivation of dry catalase by heat follows first-order reaction kinetics. The logarit,hm of the relative activit.y is proportional to the time. Typical
data are shown in Fig. 3. Thus $
= e+t,
where k, is
0
the specific rate constant. Dry heat inactivat,ion does not involve collisions between catalase and other neighboring molecules, but merely the increased vibration of intramolecular bonds. Thus it seems reasonable, t,hough not necessarily certain, that the purity of the preparation should have no effect on the inactivation rat’e. Dry heat inactivation is seen to require much higher temperatures than is visually required for solutions of enzymes. It should be noted that some of the curves of Fig. 3 do not pass through the 100% point. This is the result of insufficient drying of the samples. Initially these semiwet samples inactivate at a very rapid rate, but after some of the mater is driven off they follow the much slower inactivation rate typical of dried material. The theory of absolute reaction rates (8) gives for the specific rate co&ant kl:
where K is Boltzmann’s constant; Ir, Planck’s constant; T, the absolute temperature; R, the gas constant/mole; and AFt is the free energy
402
Il.
B.
SETI,O\J~
0 l
95 5-c. 85.5-c.
A 78 5'c. n 116 0-C
20
60
40 TIME
I
I
I
a0
100
120
IN MINUTES
of act,ivation for inactivat,ion. Thermodynamics gives AF:, in t.rrms of the heat of activntjion LIH$ and the enkopy of wtiv:Ltion A#, as AI;T z AH: - 7’ASf
(2)
11 graph of Apt, c~nlculated from the data of Fig. 3 and ICq. (I), versus absolute temperature is shown in Fig. 1. The error in these detjerminations is principally t,he tcmperaturc fluctuations in the oven, since :I 1°C. error in the o\-cn temperature is abollt, a, 0.35; (Arror in the calrulatrd valtlc of AFT. Errors in I;, are negligilk since t,his term itppears
27.600
2 27.400 \E
27.200
27.000
26.600
f?JG.
I
I
I
340
350
360
370
Temperature
'K
1. The free cnergg of actiw~tion,
,
4I ‘1, of dr\- atalnse ternperat.uw.
360
plottd
390
against the absolute
404
R.
B.
SETLOW
The values of A@ and AS obtained from Fig. 4 and Eq. (2) arc shown in Table I. For comparison there are shown similar values for heat inactivation of catalase in wat,er. Since the enzyme preparation was not pure, the latter values have dubious significance. They arc included to emphasize tjhc difference Mween the kinetics of wet and dry inactivation. Reszdts of Combined Ileuteron and Neaf Inactivation Some of t,he deuteron data, taken after the results presented in Fig. 1 were obtained, gave inactivation cross sections somewhat higher t’han
1
DEUTERONS FIG. 5. The relative
PER SQUARE CENTIMETER
activity of part~ially heat-innct,iwted rat:tlase on a lognrithmic scale VS. the number of inrident dcuterons/cm.?. 0 - 0 -, experimental points; - . - . -, resolution into t,n-o straight lines; ~~ . ~~ , curve for unheated catalnse (Fig. 1).
the 1410 A.” previously found. It was suspected that this was the resuk of partial heat inactivation of stock ratalnsc. This supposition was verified by bombarding catalase with deuterons after preliminary dry heat, inactivation (at 91%. for 30 min.) and by heat inactivation of bombarded samples. The results of these experiments were the same. The curve of per cent, activity versus deut8eron dose show~l a high init’ial slope much larger than for deut,erons alone. The data are shown in Fig. 5. The shape of the curve drawn through the experimental points ~-as the same whether the cat’alasr was heated before or after bombardment. The inactivation curve may be resolved into the sum of two straight lines (which on a linear plot is the sum of two exponenGals). The cross sections corresponding to t,hese components arc 8700 *4.” and 1410 A.‘. The latter value is found with deuteron bombardment alone. The former is greater than the geometrical cross section. If we take a literal view of the component curves of Fig. 4, \ve can say that 00$$0 heat inact’ivation yields three classes of catalase. The altered but still first is unchanged (l’i70), the second is structurally retains its cat,alyt,ic activit’y (237;) and the t,hird is completely inacbirated (607,). Drscx~ssrox
AND
I~TERPIWTITION
The dat,a for the heat inactivation of dry catalase have a simple qualitative interpretation. An increase of ent’ropy of a system is associated with a more random configuration. The denat’uration of prot,eins in solution shows an increase in entropy for the act’ivated complex (9). This more random configuration has been associat’ed with a partial unfolding of the protein molecules. From this partially unfolded activated &ate the molecule proceeds to a still more unfolded final configuration. In our experiments t’here was no evidence of a reversible reaction taking place, so that we can only infer characteristics of t’he activated state but not the final state. The decrease in entropy between the initial and activated states of dry catalase is a definite indication that the molecule is not unfolded. Beyond this, the physical significance of I he negative entropy change is not clear. One more point may be made about the process of dry heat, inactivation. The transition from ordinary stable dry cat’alase to the a&i\-ated state is not a one-step process. There exists at least one intermediate configuration which has a high relative stability. This intermediate state shows up with an abnormally high deuteron inactivation cross
-406
II. B. SETLOIV
section. It) is relevant that, three types of catulase ha\-c Iwen found by spectroscopic analysis ( 10). While it is tempting to speculate further concerning the prowss of heat, inactivat,ion, the present data do not, seem to warrant it. Two possible inberpretations of the deuteron inactivation data will be given. The first. explanation is based on t,he assumption that the molecular Iveight of catalase is about 250,000. If this is true, then an inactivation cross se&ion less t,hnn the molecule’s projected arca may mean that’ t*he molecule can only be inactivated if the energy rcleascd by a deuteron appears in certain critical regions of the molecule, or that the passage of a deuteron knocks out only half the catalyt,ic :wtivity of the molecule. A%tJ t’he higher ionization densit,ics, several primary ionizations are produced within the molecule. I~ccausc of the large mass of the deuteron compared t,o the electron clouds through which it passes, the deuteron travels along a st)raight line and the ionizutJions it produces are along this path. One might guess t’hat a deuleron must liberate energy close to one of the four hemc groups if the catalytic activity is to be destroyed. i.e., catalytic activity is not, a gootl intlicatol of catalase structure. In t)his view, a partially heat-inactivat*ccl molwl~lc would have several broken bonds distributed at random, and while still stable against inactivation by thermal energy (about l/30 e.v.), the release of about, 100 e.v. by a tlcuteron would br enough to (wry t,ho molecule to the nctivat,ed siatc anal on to an innc:t.ive configuration. The deut,cron woss section for the partially heat-inac:t.i\.at,ctl wmponent’ is larger than the molecular size. h deuteron passing ocs~sitlc t’he molecule can inactivate it,. The discrepancy may be the result’ of two factors. The events resulting from a passing deut,eron art’ not strictly confined to a lint bllt, are tlistributlerl \vithin about 3 :2. at~rmtl the deuteron’s pnt.h (7). This is simply ;L rtsult of t,hc fac:(, 1h:it, the! electric field of a charged patticlc does not, ubrupt,ly fall to zero for distances which are large compared to the particle size. If \w add 3 ;2. to all the dimensions of a cat,:tlase molecule, then t,hc ctiwtive wwu within which a deutcron may cause inact’ivation is about 7000 A.“. The steep inactivat,ion curve is obt.xincd by taking t.he diffcrcnw bct~wccn two curves (Fig. 5). The error thercforc, is larger than for 4thcr of t hc other two and may be at, least f 20’:{,. There is one important object,ion to the explanation given above. The effects of heat plus deuteron bombardment. were found for 3.70 m.e.v. deuterons. At, this energy, as remarked before, there is a 35’;/,
chance of a deuteron producing no primary ionization within a catalase molecule. Unless the effectiveness of excitations and secondary electrons is large, we would expect always to obtain an inactivation cross scction smaller than the geometrical one. We are led to a second possible int’erpretation, based on the assumpt’ion that catalytic activity is a good indicator of catalase structure. If this is true the molecular weight’ of catalase is considerably less than 250,000. If we take 3400 A. for the cross section of the molecule (see Fig. 2), then the molecular weight for the equivalent sphere is 130,000. It should be noted, however, that the theoretical curve and the experiment,al points of Fig. 2 are not in good agreement. While the heatinactivation data still give a picture of intermediate configurations preceding the activated state, the combined deuteron and heat effect can no longer be explained in terms of raising the particle of 130,000 molecular weight to the activated stat)e, since such a particle cannot have a 8800 A4.3cross section unless it is very asymmetrical (an axial ratio of about 50 to 1). This large cross section may be the result of indirect action. Under the action of heat, a catalase molecule may be in an intermediate configuration. It still has catalytic activity, but is more sensit’ive to radiation. The effect of an ionizubion within the molecular confines may now cause addiCon changes in intra,molecular forces, so that’ subsequent dissolving in water allows the molecule t,o unfold. If the molecule unfolds, oxidizing groups may now be available to act upon intact catalase molecules. Alternatively the inactivated molecule may serve as a nucleus for aggregation of active molecules. This would represent a decrease in cstalase solubility. Evidence for a decrease in soluhility of hemoglobin after deutcron bombardment has been found by Appleyard (II). In either of these cases a deuteron can miss a molecule and still inact>ivate it, and a cross section larger than that of a single molecule would be observed. It is of interest that a bacterial virus shows a somewhat similar behavior toward a combination of dry heat and deuteron irradiation 02). It is hoped that the data and discussion presented here illustrate the use of physical inactivation methods in investigation of the st,ruct,ure of large molecules. AC~NO~~LEDGME~YTS
The author wishes to thank FT. R. Adams, for his help in running the cyclotjron, nnd the other members of the Biophysics Division, for many helpful discussions.
SUMMARY
The inactivation of beef red cell cat?lase by fast dcuterons, heat, and a combination of the t,wo has been reported. The data have been analyzed quantitatively in terms of the target theory of ionizing radiation and the theory of absolute reaction rates. Evidence for the existence of at least two forms of stable catalase has been presented. The data are shown to indicate two possible alternative molecular weights (about 250,000 and 130,000) for catalase. REFERENCES
1.
LEA,
2. 3. 4. 5. 6. 7. 8.
POLLARD, E. C., Am. Scientist 39, 99 (1951). GRANICK, S., AND GILDER, H., Advances in Enzymol. 7, 305 (1937). LIVINOSTON, s., AND BETHE, H. A., Rev. Modern Phys. 9, 263 (1937). SUMNER, J. B., AA-D GRALEN, N., J. Biol. Chem. 125, :3X (1938). HALL, C. E., J. Riol. Chem. 185, 749 (1950). POLLARD, E. C., AND FORRO, F., Arch. Biochem. Hiophys. 32, 256 (1951). GLASSTONE, S., LAIDLER, K. J., AND EYRIKX, H., The Theory of R:tt)e Prowsses.
1). E., Actions of Radiations on Living Cells. .\lux~illari,
McGraw-Hill,
New York, 1947.
New York, 1941.
0. STE~RN, E. k, Advances in, Enzymok 9, 25 (1949). 10. BROWN, G. L., cited by Randall, J. T., Proc. Ro?/. Sac. (London) 11. AWLEY~RD, It. Ii., Phys. Rev. 83, 231 (1951). 12. ADAMS, w. I<., AND POLLARD, P:., Ph.+% II%v. 83, 2:SO (i!);il).
A208, 1 (1951).