932
H. FRICKE,W. LANDMANN, C. LEONEAND J. VINCENT
Vol. 63
APPLICATION OF OPTICAL ROTATION MEASUREMENTS IN STUDYING THE STRUCTURAL DEGRADATION OF r-IRRADIATED OVALBUMIN' BY HUGOFRICKE, WENDELL LANDMANN, CHARLES LEONEAND JAMESVINCENT Contribution f r o m the Chemistry Division and Biological and Medical Research Division, Argonne National Laboratory, Lemont, Illinois Received Februarg 16, lgb9
Earlier work led to the conclusion that ovalbumin treated with high energy radiations contains a broad range of protein molecules in various stages of structural breakdown. This view has now been supported by optical rotation studies on sohtions of ovalbumin after treating in the lyophilized state, under vacuum, with ?-rays. The specific levorotation [a166461 of the isoelectrically coagulable fraction and of three thermolabile fractions of increasing stability obtained from ovalbumin treated with 58.5 electronic volts per protein molecule, were found to be 60.2,47.7,42.1 and 39.5", respectively, as compared to 85.0" for ovalbumin denatured by heating a t p H 7.2 and 36.9' for the native protein. These values for the specific levorotations of the various fractions are discussed with reference t o the serological activities of these same fractions, aa measured by their reaction with antinative rabbit serum.
-
Thermal stability and serological studies2+ led to the conclusion that ovalbumin treated with high energy radiations contains a spectrum of protein molecules in which the secondary, Hbonded structure is in various stages of unfolding. The most strongly degraded molecules exhibit a characteristic coagulation reaction a t the isoelectric point of the native protein. Other molecules in more moderately injured configurations, were discovered because upon heating they are changed more easily than native molecules to the coagulable state. I n solutions of y-irradiated ovalbumin, the whole or nearly the whole loss of serological activity, as measured by the reaction of the antigen with antinative rabbit serum, was found to be associated with the coagulable and thermolabile constituents. The coagulable fraction suffered a large but not a complete loss of activity, suggesting that it is in a less strongly degraded state than protein degraded by heating. The activities of the thermolabile constituents lay intermediately between those of the coagulable and native protein and increased with increasing stability of the fractions. Measurement of the optical rotation of proteins in solution is a sensitive method for studying the disruption of their secondary structure. This paper describes the use of the method in elucidating the structural state of the protein molecules in solutions obtained from lyophilized ovalbumin that was exposed, under vacuum, to y-rays. As in earlier works, a number of fractions of decreasing structural breakdown were precipitated progressively by application of pH and heat. By measuring the optical rotation of the series of supernatants obtained in this process, we calculate the specific rotations of the precipitated fractions in the state in which they were present in the original solution. Materials and Methods The general procedures used were the same as those employed in our earlier works. I n the following, they will be described in a somewhat more detailed manner than was done before. ( 1 ) Based on work performed under the auspices of the U. S. Atomic Energy Commission. (2) H. Fricke, Nature, 169, 965 (1952). (3) H. Fricke, THISJOURNAL, 56, 789 (1952). (1) H. Fricke, C. A. Leone and W. Landmann, Nature, 180, 1423 (1957). (5) C. A. Leone, W. Landrnann and H. Fricke, "Proceedings of the Second International Conference on the Peaceful Uses of Atomic Enerey." Geneva. 1938.
The ovalbumin was recrystallized three times with NasNearly all the salt waR dialyzed off following the last crystallization and 90 t o 92% of the water, as determined by heating the protein to constant weight a t IIO", was removed by lyophilization. The lyophilized protein was placed under vacuum in glass ampules and irradiated at 0" in a homogeneous, 7-ray field a t 1.5 X 106 rad./hr. Radiation dosage was determined with the ferrosulfate dosimeter (G[Fe+++] = 15.5) and expressed in terms of electron volts absorbed per protein molecule (e.v./mol.). In this calculation, we used 45000 as the molecular weight of ovalbumin and 7% H, 50% C, 15.5% N, 26% 0, 0.1% P, 1.6% S as its elementary composition. Photoelectric and recoilelectric absorption values were obtained from Lea.' Irradiated ovalbumin is stable if it is kept in the lyophilized form and under vacuum at 0 " . When placed in solution its relative instability toward temperature and p H (Fig. 1) must be kept in mind during solvation and in storing and testing it. To avoid or reduce secondary degradation, all initial processing was carried out near 0", and as close t o neutral p H as possible. Owing in part to crosslinkage, and in part to structural unfolding, irradiated protein is more difficult t o dissolve than native. I t dissolves at an impracticably slow rate a t neutral p H . Dissolution is hastened a t both acid and basic pH values and since the coagulable constituents are insoluble in the acid region PH 3.5 to 6, dissolving the protein was carried out a t basic pH. I n the dosage range used, up to 120 e.v./mol., the irradiated protein went rapidly into solution a t pH 9. The irradiated powder was added a few milligrams a t a time to water adjusted to p H 9 with NaOH. The solutions were prevented from dropping below pH 7 by the periodic addition of small volumes of 0.1.N NaOH. The dissolution process required about 90 min. Tests showed that dissolved protein treated with 58.5 e.v./ mol. could be kept for two hours at pH 9 without noticeable increase in structural degradation. After being dissolved, the irradiated protein was brought to H 7.2 and traces of insoluble material removed by centriggation. At this pH and 0", the solutions of irradiated protein were stable for several hours, during which period the optical rotation was measured. Ovalbumin is fragmented under the influence of ionizing radiations, but this effect was small enough in our work, to not cause a significant rise in non-protein nitrogen as determined by the micro-Kjeldahl procedure. Therefore, total nitrogen determinations on the whole irradiated systems or on fractions derived from them were converted t o protein by using the conversion factor, 6.45, of the native ovalbumin. Fractionation.-(a) The coagulable fraction of a solution of irradiated protein was obtained by lowering the pH with 0.1 N HCl to 4.85, which is the isoelectric point of native ovalbumin. The coagulation rocess went t o completion within 10 to 20 minutes. T l e supernatant, after centrifugation, remained clear for a few hours, showing that the coagulable and thermolabile fractions could be cleanly separated from each other. Further tests revealed ( 6 ) R. A. Kekwick and R.
K. Cannan, Biochem. J . , 80, 227 (1936). (7) D. E. Lea, "Actions of Radiations on Living Cells," Cambridge University Press, 1947, p. 347.
STRUCTURAL DEGRADATION OF 7-IRRADIATED OVALBUMIN
June, 1959
OPTICAL
TABLE I ROTATION MEASUREMENTS ON FRACTIONATED SOLUTIONS OF ?-IRRADIATED OVALBUMIN Obsd.
Dosage, e.v./mol.
0 15.0 30.3 58.5 90 120
933
--Relative
concn.-
D
LI
0.062 ,123 .a5 ,355 .51
0.21 .24 .28
Lz
0.21
I 36.9 40 3 42.1 47.1 51.7 57.1
SI
[~Y]~6461--7
s2
37.0 36.7 40.8
39.0 40.0 43.4 47.2 50.9
that no additional coagulate was formed when pH of the supernatant was varied with HCI and NaOH over a broad range, pH 3 to 6, proving that pH 4.85 was close enough to the isoelectric point of all coagulable constituents to secure their complete precipitation. ( b ) After the roagul:tble fraction had been removed, two fractions of theimolal,ile constituents were precipitated successively by exposing the supernatant to 49.25' for 6 lir. and 62.05" for 4 hr. a t pH 7.2. Aft'er the first heat treatment the supernatant was adjusted to pH 4.85 with HCI. The precipitate t,hat formed was removed and pH of the new supernatant Rolution readjusted to pH 7.2, with NaOH and then the process was repeated at the higher temperature. The native protein is practically stable a t 49.25", pH 7.2, so the fraction of irradiated protein removed at. this temperature contained no appreciable admixture of nat)ive protein. Terminology.-Solutions of native and iri-adiaLed protein are called N and I, respectively. The coagulable fraction of I is called D and the labile fractions separated successively by the use of any particular procees are called L1, L,, La,etc. The series of supernatants obtained in the progressive removal of these fractions, are called SI,S2,Sa,S1,etc. The concentrations of D , L 1 , Lf, La, etc., calculated as fractions of the protein concentrations of I, are called [ D ] , [ L ] , I L l , [.L1,et?. Optical Rotation .-Measurements a w e made with a Rudolph precision polarimeter. graduated to 0.01 degree, using the mercury line 5461 A. and 1 and 2 dm. tubes. The measurements were made in a cold room near 0".
-
D redis-
Sa
solved
60.9 59.0 61.4 69.9
39.5
...
--Calcd. D
61.2 57.0 58.3 61.3 63.1
- [al%m-L1
L2
46.2 449.2 447.7
42.1
50 W'
I-
3
40
3 W
a
0
o
30
W
W
;I
20
Z W
0
U
w
a
IO
I
I
I
8
9
IO
PH. Fig. 1 .-Structural degradation of -&radiated ovalbumin exposed for 3.5 hours to NaOH a t different pH values; radiation dosage 58.5 e.v./mol. The native protein is stable in this pH range. The coagulate was collected a t pH 4.85; temp. 0".
coiicentratioiis of salt did not affect the optical Experimental Results rotation. Hence, we may calculate the specific The data on the optical rotation of solutioiis of levorotation for D, in the structural state in which irradiated ovalbumin are presented in Table I. it exists in I, from eq. 1. For the native protein the specific levorotation, IDI[aDI ( 1 - [ D l ) [ ~ i= l [a11 (1) - [ a I 6 5 4 6 1 , obtained was 36.9', which agrees satisfactorily with the results of earlier w0rkers.~~9where [all, [as,]and [ a D ] are the specific levorotaThe y-irradiated protein has an increased specific tions of I, SI and D, respectively. The values of levorotation, which varies linearly with radiation [QID]thus calculated are shown in Table I. They lie dosage. A linear relationship is found also for the in the range of 57.0 to 63.1' and have a mean value of 60.2'. They show no apparent dependence on variation of [D] with radiation dosage. The table shows that the increased levorotation radiation dosage. In order to measure the optical rotation of D of I is, in part but only in part, associated with the coagulable constituents, the levorotation of SI directly, the coagulate obtained on shifting pH being lower than that of I, but higher than that of of I to 4.85 was centrifuged off, carefully washed K. Some of the isoelectrically soluble coiistitueiits with pH 1.85 HCI and then redissolved in pH 9.0 NaOH. The values of [ a ~obtained ] from the soluof I must possess a relatively high levorotation. Since tests showed that the specific rotation of I tions of these dissolved coagulates are given in did not depend on the protein concentration used Table I. They agree well with the calculated in its measurement, we may assume that the values, indicating first that the assumptions used rotation of I is the sum of the rotations of its in deriving eq. 1 are valid and, second, that D various fractions. Furthermore, the operations can be precipitated and redissolved without altering used in removing D mould not be expected to its molecular structure. In order to establish the role played by the affect the rotation of the soluble constituents of I. As a result of the manipulation of pH of I to 4.85 thermolabile constituents in the increased levoroand the subsequent readjustment of SI to pH 7.2, tation of SI over native ovalbumin, the thermal the latter solution contains somewhat more fractionation procedure was applied to the samples NaCl than I, but tests revealed that such small that had absorbed 15.0, 30.3 and 58.5 e.v./mol. The second heat-treatment was applied only to (8) H. F. Holden and h l . Freeillan, Australian J . R r p . B i d . M e d . , protein treated with 58.5 e.v./mol. The optical 7 , 13 (1930). rotahions thnt vere determined for the several (9) H. A. Barker, J . B i d . C h o n . , 103, 1 (1933).
+
931
H.
FRICKE, W. LANDMANN, C. LEONEAND J. VINCENT
supernatants are given in Table I. The results show that the increased levorotation of the several SI'S over native ovalbumin is associated wholly or mainly with the thermolabile constituents, their removal causing the optical rotation to approach that of the native protein. In the samples that adsorbed the two lower dosages of radiation, the specific levorotations of the Sz solutions were not significantly different from that of native ovalbumin showing that the increased levorotation of the S1 solutions was essentially derived from the Ll's. In the sample that absorbed 58.5 e.v./mol. the removal of even the Lz fraction did not completely restore the levorotation of the Soto that of native ovalbumin. At this relatively high dose of radiation there may be no appreciable amount of structurally uninjured protein left in I. In order to draw quantitative conclusions from these experiments n e must assume that the heatings did not affect the isoelectrically soluble constituents remaining in the supernatants of the heated systems. Strict proof cannot be given, but supporting evidence is provided by the fact that for the smaller dosages, removal of the thermolabile constituents brought the optical rotation of S1 back to that of N. Further support is given by the fact that the thermal conversion of native ovalbumin to the coagulable form is a first-order r e a c t i ~ n . ~ ~This ~ ~ -indicates '~ that a t least for the native protein the degradation of the molecular structure by heat is an all-or-none reaction, which is not accompanied by structural changes in the soluble molecules. Direct evidence that heating native ovalbumin leaves the isoelectrically soluble protein molecules unaltered was obtained earlier from serological ~ t u d i e s . ~We have now further verified this conclusion by means of optical rotation measurements. Solutions of native ovalbumin a t pH 7.2 were heated a t several selected temperatures between 62.0 and 71.0' so as to convert various proportions of the protein into the coagulable form. The altered protein was coagulated by adjusting the systems to pH 4.85. The specific optical rotations of the cleared supernatants were measured. No significant changes from the value for the original native protein were obtained. Although the question whether the thermal degradation of irradiated ovalbumin is an all-ornone reaction remains undecided, we shall tentatively assume that this is the case. We may then calculate the specific levorotation [ o ( L ~ ] of the thermolabile fraction Ll from an aiialog of eq. 1
+
[L,I [ a ~ i I (1
- [Dl - ILlI) Iad
= (1
- [nl)[W3il (2)
A similar equation was used for calculating the specific levorotation [CIL,~ of Lz. The values of [ C Y L ~in ] Table I show no dependence upon radiaThe tion dosage. The mean value is -47.7'. value for the one calculated [CUL,]is -42.1'. (10) H. Chick and C. J. Martin, J. P h y s i o f . (London), 40, 404 (1910). (11) H.Chick and C . J. Martin, ibid., 4S, 1 (1911). (12) H. Chick and C. J. Martin, ibid., 45, GI (1912). (13) H . Chick and C. J. Martin, KolEoidchem. Bezh., 6, 49 (1914). (14) P. S. Lewis. Floehem. J . . 20, 9G5 (192G). ( 1 5 ) H.K. Cubin, zbzd., SS, 25 (1929).
Vol. 63
Discussion We showed earlier4t6that the loss of serological activity of solutions of yirradiated ovalbumin, as measured by the reaction of the antigen with antinative rabbit serum, was associated wholly or nearly wholly with the coagulable and thermolabile fractions. The coagulable fraction suffered a marked but not a complete loss of activity, the residual activity being about 16% of that of the native protein. The losses suffered by the thermolabile fractions were smaller and decreased with increasing stability of the fractions. It was suggested that not only the coagulable fraction but also the thermolabile fractions are in states of partial structural unfolding, the extent of which in any particular fraction increased with decreasing stability of that fraction. The fact that the coagulable fraction retained an appreciable amount of activity, indicated that it had not undergone complete unfolding. Disruption of the H-bonded secondary structure of proteins with the resultant unfolding of the polypeptide chain has been found invariably to lead to a large increase in levorotation of solutions containing them.s,9,16,17,17a The effect has been discussed theoretically by Fitts and Kirkwood. 18v19 The increase in levorotation depends on the denaturing agent used. Holden and Freemans measured the levorotation of ovalbumin subjected to a variety of treatments resulting in coagulation at the isoelectric point and iound values in the range of 58 to 100' (at 5461 A.). These different values might at least in part reflect differences in the extent of unfolding obtained in the different cases. The increased levorotation values found for the various fractions studied in irradiated ovalbumin supports the conclusions as to structural unfolding drawn from the serological evidence. The levorotation of the coagulable fraction lies in the lower range of values found in Holden and Freeman's study, suggesting that the molecules of this fraction are not in a completely unfolded state. The TABLE I1 COMPARISON OF OPTICAL ROTATION A N D SEROLOGICAL ACTIVITYO F DIFFERENT FRACTIONS OF A SOLUTION OF ?-IRRADIATED OVALBUMIN.RADIATION DOSAGE 58.5 e.v./ MOL.
Fraction
Relative concn."
- [CZ~~KMI
Serological activity
Relative degree of unfolding in terms of Serological Optical rotation activity
D 0.25 58.3 0.17 1 1 I,, .28 47.7 .51 0.50 0.59 L* .31 42.1 .77 0.24 0.28 .25 39.5 .80 0.12 0.13 Sa N 36.0 1 0 0 a With reference t,o concentration of protein in I. (IC,) P. Doty and E. P. Geiduschek in: H. Neurat,h and 1C. Bailey, "The Proteins," Academic Press, New York, N. Y . , 1953, Vol. la, p. 393. (17) C. Cohen, Nature, 176, 129 (1955). (17a) 5. A . Schellman, Compl. Rend. Trau.Lab. Carlsberg, S6r. chim., SO,3G3 (1956-58). (18) D. D. Fitts and J. G. Kirkwood, J . Am. Ckem. Soc., 78, 2650 (1956). (19) D. D. Fitts and J. G . Kirkwood, Proc. Watl. A c . Sei. (U.S.), 4a, 33 (195~).
EFFECT O F tu-RADIATION
June, 1959
levorotations of the different thermolabile fractions lie intermediately between the values for the coagulable fraction and the native protein and decrease with increasing stability of the fraction. In order to show the correlation with the serological activities, we present in Table 11, for the radiation dosage 58.5 e.v./mol., the specific levorotations of the different fractions, taken from Table I , and the relative serological activities measured on the same preparations.6 To compare these two sets of data, we calculate the quantity ([‘J‘FI
-
[aNI
)/( [an]
-
[aNI
ON
AQCEOUS GLYCINE
935
and the quantity obtained when the [CUI’S are replaced by the corresponding values for the serological activities. (F denotes any particular fraction.) These two quantities may be described as representing the relative degrees of unfolding of the fraction F, in terms of levorotation and serological activity, respectively. The correlation between the two sets of values is seen to be quite good. Although the meaning of this comparison is not, wholly clear, it is felt that it gives further support to the view that the relation of the serological activity to the extent of unfolding in the various fractions is a causal one.
THE EFFECT OF U-RADIATION ON AQUEOUS GLYCINE BY CHARLES R. MAXWELLAND DOROTHY C. PETERSON Department of Health, Education and Welfare, Public Health Service, National Institutes of Health, National Cancer Institute, Radiation Branch, Bethesda, M d . Received February 16, 1959
Oxygen-saturated and oxygen-free 1 11.1 solutions of glycine have been irradiated with a-particles from an exterior Po210 HCOCOOH and HZOZare reported as a function of dose. The relative yields of these source. Yields of HCHO, “I, compounds are quite different from those previously reported for X-rays. The specific yields of these products are greater, particularly in the oxygen-saturated solutions, than would be predicted by an indirect free radical mechanism in the bulk of the solution.
Introduction The experiments described here on the radiolysis No concerted effort has been made to determine of aqueous glycine with tu-particles from Po21ohave the effect of the rate of linear energy transfer (LET) been restricted to the measurements of the major upon the radiolysis of aqueous glycine. The effect products at small doses. Where possible the yields of X-rays has been studied by Dale, Davies and have been determined as a function of dose and the Gilbert,’ Stein and Weiss12Barron, Ambrose and initial yields evaluated as the slopes of the plots of Johnson,a and Maxwell, Peterson and S h a r p l e ~ s . ~these data. The effect of electrons upon the system has been reExperimental ported by Maxwell, Peterson and White5 who a-Particles were introduced into the glycine solution showed that the yields of the products were the through a mica window submerged in the solution. The polonium source was a thin spot of metal approximately 1 same for 50 Kv. X-rays a t a dose rate of 1 X 1020 cm. in diameter on the end of a g / ~ a ” tantalum rod housed e.v./l. min. as for 150 Kv. electrons a t a dose rate of in a tight fitting glass tube closed a t one end with a mira m1.6 X loz3e.v./l. min. However, the LET for window. This window was between 1.0 and 1.2 mg./cm.* these two irradiations are essentially the same. thick and was sealed to t h e glass with black wax. The rod could be moved so that the polonium was in Dale, Davies and Gilbert6 have reported the yield of tantalum contact with the window during an irradiation. The source NH, from alkaline sir-saturated solutions of glycine and housing were completely inside the sample handling and irradiated with the recoil particles from the BIO (ntu) degassing system so that there was never more than a few Li7 reaction. However only very large doses of millimeters pressure differential across the window. The all-glass apparatus consisted of a degassing chamber the order of 1.5 x loz5e.v./l. were used and only and an irradiation chamber. These chambers were conthe yield of NH, was determined. Weeks’ has stud- nected to each other and to a vacuum line in a manner which ied the effect of 30 MeV. He ions upon aqueous gly- allowed individual evacuation and the transfer, by gravit,y, cine but his experiments were designed primarily for of solution from the degassing chamber to the irradiation the detection and estimation of compounds pro- chamber. For gas-free vacuum runs the solution to be irradiated was duced in low yield. The reported yields a t the degassed by boiling under the low pressure obtained with a very large doses necessarily used probably do not mechanical pump. The solution was placed in the degassing represent the initial yields of these products before chamber where it was stirred violently by a Teflon bar for 20 minutes during which time it was opened frequently for the onset of secondary reactions. short periods to the vacuum line for the removal of evolved (1) W. A I . Dale, J. V. Davies and C . W. Gilbert, Bioclrem. J . , 46,93 ( 1 949). ( 2 ) G. Stein and J. Weiss.
.I. Chem. Soc., 3256 (1949). (3) E. S. G . Barron, J. Ainbrose and P. Johnson, Radiation Research, 2, 145 (1955). (4) C. R. Maxwell, D. C. Peterson and N. E. Sharpless, ibid., 1, 530 (1954). (5) C. R. Maxwell, D. C. Peterson and W. C . White, ibid., a , 431 (1955). ( G ) W. M. Dale, J. V. Davies and C. W. Gilbert, Eiochem. J . , 46, 543 (1949). (7) B . A t . Weeks, University of California Radiation Laboratory Report DCRL-3071 (July 1955).
gases. The irradiation chamber was evacuated with the same vacuum line and then swept free of any last traces of gases with water vapor from the degassed solution. Sample was drained from the degassing chamber into the irradiation chamber until the mica window was submerged approximately 3 mm. The sample was stirred gently during the irradiation by another Teflon bar rotating a t 120 r.p.m. After the irradiation the sample size was determined by weight. Sample size varied from 15 to 25 ml. depending upon the radiation chamber used. For oxygen saturated runs, the entire system was evacuated and the solution partially degassed. Oxygen was admitted to atmospheric pressure. The solution was then
