Marked change of circular dichroism of l -cystine solution with temperature

BIOCHIMICA ET BIOPHYSICA ACTA BBA

36036

MARKED SOLUTION

CHANGE WITH

OF CIRCULAR

DICHROISM

OF L-CYSTINE

TEMPERATURE

TOSHIO T A K A G [ * AND N O B U T A K A ITO**

Institute .for Protein Research, Osaka University, Kita-ku, Osaka ( J a p a n ) (Received J u n e 29th, 1971)

SUMMARY

Circular dichroism of L-cystine solution was studied between - - 7 °° and 8o °. The CD spectrum changed markedly with temperature. The negative CD band at 255 nm assigned to the disulfide chromophore increased its intensity with decrease of temperature. The positive CD band at 220 nm assigned to carbonyl chromophore showed less significant temperature dependence. Another negative CD band at 200 nm showed temperature dependence comparable to that of the band at 255 nm. The observed marked temperature dependence of CD of the disulfide chromophore was discussed in relation to the possible equilibrium between the two conformations of the disulfide group which are mirror images.

INTRODUCTION

Disulfide groups are known to prefer either ot two skewed, dissymmetric conformations which are mirror images of each other and have a dihedral angle of about 9 oo (Fig. I)2, 3. Such a conformation is predicted for the general case of compounds with the following s t r u c t u r e , - A - B , having four unshared electrons in p-orbitals of adjacent

,-s. i (M)

(P)

Fig. I. Two possible conformations of the disulfide group. T h e y are mirror images, and will be tentatively n a m e d M (minus) and P (plus) forms, respectively, according to the helicity rule of (,'~AHN et al. ~, Abbreviations: Conformations of disulfide groups with minus and plus helicity will be represented by the symbols, M a n d P, respectively, according to the helicity rule of CAHN et al. 1 (see Fig. i). * To w h o m reprint requests should be addressed. ** On leave of absence from N a r a Technical College, Nara (Japan), Present address : Ministry of Education, Tokyo (Japan).

Biochim. Biophys, Acta, 257 (1972) 1-Io

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atoms 4. I f group R shown in Fig. I is symmetrical, the two contbrmers have equal stability, and the compound consists of a racemic mixture of disulfide groups with conformations P and M (see Fig. I). However, the disulfide group of L-cystine is linked to an asymmetric carbon with the L-configuration through a methylene group on both sides. Therefore, the L-cystine molecule with either of the two disulfide conformations is not a mirror image of the other. Construction of a space-filling molecular model shows that the L-cystine molecule with the disulfide group in the P conformation differs from that with the disulfide group in the M conformation in the mutual spatial arrangement of the groups attached to both ends of the disulfide group. Presumably the two conformations differ in their stability. There are m a n y data suggesting that the difference in stability between the two conformations is not big. In the L-cystine crystal, disulfide groups have the P conformation 5. On the other hand, in crystals of L-cystine" 2HC1 (ref. 6,7), L-cystine- 2 H B r (ref. 8), and N,N'-diglycyl-L-cystine dihydrate 9, the disulfide groups have the M conformation. In the hen's egg-white lysozyme molecule which has four disulfide groups, two of them have the P conformation, and two have the M conformation 1°. I f either of the two conformations were preferred due to a big difference in stability, such diversity would not be expected. In solution both of the two conformations are expected to be present in equilibrium without a large excess of either. However, the equilibrium in solution has not been studied. It is to be noted that TOENNIES AND LAVINE11 reported in 193o a marked temperature dependence of [a~D of L-cystine solution. Their result has been cited by DJERASSI 2 a s suggesting a shift of equilibrium between the two conformations with temperature. SZANTAY et al. 12 also interpreted the temperature dependence of [alp of anomers of di-5-(2'-deoxyuridilyl)disulfide as reflecting a shift of equilibrium between two conformers differing in their disulfide conformation. Itowever, the viewpoint that one of the two conformations is predominant in a solution is the prevailing one (see, for example, ref. 13). Measurement of temperature dependence of the CD spectrum of L-cystine solution seemed essential to make the situation clear, because attention could be focussed on the disulfide chromophore. Curiously, no attempt has been made in this line. In this paper, we want to show a marked temperature dependence of the CD spectrum of L-cystine solution that m a y be correlated with an equilibrium among the conformers in solution.

EXPERIMENTAL

Reagents L-Cystine was recrystallized according to the method of OUGHTON AND HARRISON5. Ethanol was spectroscopic grade. Guanidine hydrochloride was recrystallized according to the method of NOZAKI AND TANFORD14. Other reagents were of reagent grade and used without further purification. Circular dichroic and absorption spectra A Jasco J - I o CD/UV recorder was used. The CD intensity was calibrated using an aqueous solution of D-Io-camphorsulfonic acid assuming Ae =: 2.20 ° .cm ~ .dmole -1 at 291 nm. To calculate molar ellipticity, [0] -- 3300 (eL -- eR) was used, where Biochim. Biophys. Acta, 2 5 7 (1972) I - I O

CIRCULAR DICHROISM OF CYSTINE

3

(eL -- eta) is the difference between the molar extinction coefficients for left and right circularly polarized light. The temperature of the sample cells was controlled using three kinds of constant temperature apparatus. (I) A small brass muffle obtained from Jasco was used to house I-, 2-, 5-, or I o - m m square cells. Its thermal insulation was improved by surrounding it with an ebonite plate. An inlet of d r y N 2 was provided to prevent fogging. Measurements could be made using this muffle between - - 2 o ° and 9 o°. (2) Another muffle was made from a brass block. Cells with o.I or o.2 m m length could be housed in a square hole. T h e r m a l insulation was provided b y an ebonite plate. An N 2 gas inlet was provided. The possible range of measurements was the same as above. These two pieces of apparatus were thermostatically controlled b y circulating water or ethanol at a constant temperature from a H a a k e NBe or a H a a k e KT-62 constant-temperature bath. Sample temperature was determined by measuring temperature of water or ethanol in the baths and reading the former on calibration curves drawn in advance b y measuring temperature of sample cell and bath. A precision t h e r m o m e t e r model T-oo 5 (Takara Thermistor I n s t r u m e n t Co.) was used. (3) For measurements below - - 2 o °, a specially designed apparatus was used. Its outline is shown in Fig. 2. Measurements were mostly carried out using a dry ice-acetone mix-

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Fig. 2. Diagramatic side-view of the apparatus for low temperature measurements. (I) Stainless steel inner cylinder to house cooling medium; (2) thermocouple; (3) air drainage; (4) stainless steel outer cylinder; (5) cell compartment of the CD/Ultraviolet recorder; (6) cell; (7) window; (8), copper cell holder. ture as the cooling medium. W h e n the mixture had been removed, the temperature increased very slowly due to the high heat capacity of the apparatus. Thus, measurem e n t could be made between - - 7 °0 and room temperature continuously. The curve of molar ellipticity against temperature thus obtained agreed well with d a t a obtained using Method I between - - 2 0 ° and 0 °. Square cells with a p a t h length of I - I 0 m m could be used. A bent glass tube was fused on an ordinary square cell, and the end was in every case sealed after loading of the sample solution to make installation in a Biochim. Biophys. Acta, 257 (1972) i-IO

4

T. TAKA(;I, N. 11"()

vacuum possible, l:ogging could be prevented by evacuation of the space between the outer and inner cylinders (see Fig. z). The temperature of the sample was measured by a copper-constantan thermocouple installed in the cell holder. The electromotive force was read by a Hitachi model QPDa4 recorder. Changes in wflume of the solution were corrected using the expansion coefficient of the solvent when necessary. i

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RESULTS

We have measured the CD spectra of L-cystine in o.I M HCI* down to 19o mn at various temperatures, and typical examples are shown in Fig. 3a. The spectra have negative CD bands at 255 nm and positive ones at 22o nm. Shoulders were observed around 2oo nm. The observed spectra had apparent mininla around 19o nm. However, 19o nm was the limit of the performance of our instrument under the conditions of the present experiments*" and we have no confidence in the result below 19o nm. As shown in Fig. 3b, the measurement was extended down to --7 °0 bv the addition of ethanol. The presence of a negative CD band around 20o nm became more evident. Addition of ethanol decreased the intensity of the longest wavelength band and possibly that of the band at 2oo nm. No appreciable effect was observed on the band at 22o nm. The marked temperature dependence shown in Figs. 3a and 3b is to be noted. We measured the absorption spectrum of L-cystine in o.I M HC1 at 4 ° and 62 °. No change comparable to that observed with the CD spectra was detected around 255 nm, except a slight sharpening at 4 °. The CD spectra were essentially independent of the concentration of L-cystine between o.6 and 5 mg/ml as far as the longest wavelength CD band was concerned, The conditions required prevented the variation of concentration in the far ultraviolet region. Fig. 4 shows the effect of temperature on the near ultraviolet CD spectra of L* T h e m e d i u m w a s s e l e c t e d b e c a u s e t h e d i s u l p h i d e g r o u p is s t a b l e in a c i d i c c o n d i t i o n s a n d t h e i o n i z a b l e g r o u p s a r e e x p e c t e d t o b e c o m p l e t e l y , p r o t o n a t e d . M e a s u r e m e n t of t e m p e r a t u r e d e p e n d a n c e in o . i M HC1 g a v e a c u r v e i d e n t i c a l t o t h a t in Fig. 5. ** A c c o r d i n g t o t h e s u g g e s t i o n o f t h e m a n u f a c t u r e r w e d i s c a r d e d r e s u l t s o b t a i n e d w h e n t h e a p p l i e d v o l t a g e t o t h e p h o t o m u l t i p l i e r e x c e e d e d 65o V.

Biochim. Biophys. Acta, 257 (r972) 1 - 1 o

CIRCULAR DICHROISM OF CYSTINE

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Fig. 4" CD spectra of, (a) L - t r y p t o p h a n (0.95 mg/ml) and (b), L-tyrosine (o.28 mg/ml) in o.i ~[ He1 in the presence of 9o% (v/v) ethanol at 20 ° ( - - ) and --7 °0 (-- -- --). P a t h length, IO ram.

t r y p t o p h a n and L-tyrosine in aqueous ethanol. Comparison of Figs. 3 and 4 clearlv shows that the temperature dependence of L-cystine is distinguished. Fig. 5 shows the plot of CD intensity of the band at 255 nm measured in the same conditions as in Fig. 3, At higher temperature, both of the two curves became flat, It is to be noted that the curve in aqueous ethanol became slightly concave below - - 4 °0 . The reliability of measured temperature in the region is critical in order to judge the credibility of the concavity. Temperature was usually measured by plugging the probe junction of a thermocouple in the brass block in direct contact with the sample cell. The temperature difference between the brass block and the sample solution was directly measured by setting the other junction in the cell filled with silicon oil in vacuum. The difference was found to be less than 2 ° at - - 7 o°. The observed curvature is, therefore, credible, O

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Fig. 5, The t e m p e r a t u r e dependence of the molar elliptieity at 255 n m of L-cystine in o.i M HCI (Curve i) and in w a t e r - e t h a n o l m i x t u r e (I :9 (v/v)) containing o.I M HC1 (Curve 2). Values denoted b y crosses above 20 ° were obtained b y lowering t e m p e r a t u r e from 8o °, and those below 20 ° were o b t a i n e d b y raising the t e m p e r a t u r e from - - i o ° (curve I) and --7 °° (curve 2), respectively. D o t t e d lines indicate possible u p p e r a n d lower limits of extrapolation. Squares indicate d a t a obtained at 20 ° and at t e m p e r a t u r e of liquid N~ in the water-glycerol mixture. For details, see text.

Biochim. Biophys. Acta, 257 (I972) i - i o

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T. TAKAGI, N. ITO

I f the observed temperature dependence reflected a shift of equilibrium among conformers, Curve 2 in Fig. 5 was expected to flatten at lower temperature. The observed concavity below - - 4 o° suggested the beginning of flattening. I f a somewhat daring extrapolation is permitted, the curve m a y flatten to give a value between -- 44oo and -- 55oo °- cm 2- dmole 1 as shown in Fig. 5- To confirm that the CD intensity at 255 nm further decreases by lowering the temperature, the intensity was measured at the temperature of liquid N 2 using a water-glycerol (I :x, v/v) mixture as solvent. Plunging the solution, in a cell of x m m light path mounted in the inner cylinder, into liquid N 2 gave a clear glass. Then the inner cylinder was promptly installed in the outer cylinder, and the intensity at 255 nm was followed. The glass became opaque, and had cracks within 5 min in most cases. Two data obtained with the samples that remained clear for a relatively long time and gave a plateau on the chart (intensity v s . time) were included in Fig. 5. They settled in the range of possible extrapolation of Curve 2, though this could be fortuitous. Fig. 6 shows the temperature dependence of the intensities of the three major CD bands. I t is to be noted that the temperature dependence was significant and of the same order for the bands at 2oo nm and 255 nm. The CD band at 22o nm was less affected by the change of temperature than the above two CD bands. FIESER 15 has suggested that two intramolecular hydrogen bonds can be formed only for the L-cystine molecule with P conformation, and thus stabilize the conformation. I f so, and the temperature dependence of the CD of L-cystine solution reflects a shift of equilibrium among conformers, it was expected that the presence of concentrated guanidine hydrochloride would destabilize the possible hydrogen bonds, and thus affect the temperature dependence. As shown in Fig. 7, 6 M guanidine showed no appreciable effect on the temperature dependence of the CD of L-cystine solution.

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Biochim. Biophys. Acta, 257 (1972) i - I O

CIRCULAR DICHROISM OF CYSTINE

7

DISCUSSION

As shown in Fig. 3, the CD spectrum of L-cystine in o.I M HC1 has a negative CD band at 255 nm, a positive one at 220 nm, and a shoulder at 20o nm. Major features were not affected by the presence of 90% (v/v) ethanol. The obtained results at 20 ° are in agreement with those reported by VELLUZ AND LEGRAND 16, BEYCHOK 3, and COLEMAN AND BLOUT13 except in the region below 21o nm. The CD spectrum below 21o nm has only been reported by VELLUZAND LEGRAND16. They observed a negative CD band at 195 nm. COLEMANAND BLOUT13 presumed the presence of a negative CD band between 18o and 19o nm by analysis of their optical rotatory dispersion data. Presence of a negative CD band at 200 nm was first confirmed in the present study. Its presence became more evident by the measurement at - - 7 °0 (Fig. 3b). The obtained spectra suggest that there is at least one more negative CD band at or below I9o nm. Since the data below 19o nm are not reliable, no further discussion will be made about the details of the spectra below 19o nm. The CD band at 255 nm has been confidently assigned to the disulfide chromophore by COLEMANAND BLOUT13. As will be described later, the real character of this CD band is a controversial problem, and the discussion is not yet settled. This situation makes the discussion of the present results extremely difficult. We are forced to stop discussion at a tentative step. We rather hope that our present results will contribute to the solution of the problem. The CD band at 220 nm has been assigned to a n-~* transition of the carbonyl group lz. We observed that cysteine and alanine also showed a similar positive band at 21o nm. The newly observed CD band at 200 nm showed a similar temperature dependence to that of the CD band at 255 nm assigned to the disulfide group (Fig. 6). The CD band at 220 nm assigned to carbonyl group showed less marked temperature dependence than the above CD bands. I f the temperature dependence of the CD band at 255 nm is intimately related to the conformation of the disulfide group, the common behavior with respect to temperature may favor assignment of the CD band to a disulfide transition. The marked temperature dependence of CD spectrum of L-cystine solution is the most significant observation in the present study. Temperature dependence was marked with the CD bands at 255 nm and 200 nm. Since we have detailed data of the temperature dependence only with the former, discussion will be confined to it. The temperature dependence of the CD band at 255 nm shown by Curve 2 in Fig. 5 shows a sigmoidal shape, though the extrapolation to lower temperature is arbitrary. Such a curve is expected for an equilibrium mixture of conformers with different optical activities in which population changes with temperature. With an equilibrium mixture of conformers of a small molecule such as L-cystine, the more stable conformer(s) become predominant with decrease of temperature, and each conformer becomes more equally abundant with increase of temperature. Curve 2 in Fig. 5 levelled off at higher temperatures. This suggests that the tendency to be more equally populated comes to completion in the temperature range. As shown in Fig. 7, 6 M guanidine hydrochloride had no effect on the temperature dependence of the intensity of CD band at 255 nm. The result suggests that the difference in stability among conformers is determined not by intramolecular hydrogen bonds but by steric factors. Now we must confront a serious problem. Namely we must know what kind of Biochim. Biophys. Acta, 257 (1972) I-IO

T. T A K A G I , N. IT()

confl)rmational change is important in determining tile feature of tile CD band at 255 nm. As shown in Fig. I, the cvstine residue is a unique chromophore in that it has an inherently dissymmetric structure in the disulfide group. All other chromophores of various amino acid residues are symmetrical, and their optical activities are induced by the interaction with transitions of other chronaophores and bv tile perturbation due to the asymmetric environment produced by the a-carbon atom. The optical activity of tile CD band at 255 nm of L-cystine consists, therefore, of tile intrinsic optical activity due to the inherent dissymmetry of the disulfide chromophore and the extrinsic one induced by peripheral effects. Which of the two components is predominant in the observed CD spectrum is a controversial problem at the present time. COLEMAN ANJ) BLOUTla suggested that the sign and intensity of the CD band at 255 nm is dominated by peripheral effects rather than by the effect of the helicity of disulfide group in the cystine residue. Recently, LLXDERBERG AND MICHL17 also laid stress on the peripheral effects in their theoretical study on the optical activity of organic disulfides. They considered that the first CD band is the product of overlap of two CD bands with opposite sign and almost equal intensity. They supposed that each of the two CD bands is dominated by the intrinsic optical activity, but the peripheral effects became apparently predominant clue to the cancellation of the two bands. Their calculation was carried out with H2S 2 with various fixed conformations and, therefore, the obtained results can only be applied to actual optically active disulfide compounds with reservations. Thus, the present knowledge about the nature of the first CD band of L-cystine is quite indefinite. A few comments can be made on the above problem based on the results of present study. Tile temperature dependence of the CD spectrum of a L-cystine solution must reflect a shift of equilibrium among possible conformers differing in their optical activities. Peripheral effects are determined by the mutual orientation between the disulfide group and group(s) attached to the asymmetric carbon atom. They are linked through single bonds. Their internal rotations are presumed to be less restricted than that of tile bond between the two sulfur atoms of which barrier to rotation has been estimated at between 5 and I5 kcal/mole 18-'~, and less likely to be affected by temperature change within the range of present study. Supporting data, though indirect, are obtained by tlle measurements of the effect of temperature on the CD spectra of L-tyrosine and L-tryptophan. CD spectra of the symmetrical aromatic chromophores of these compounds in the near ultraviolet region are indeed the products of peripheral effects. It is to be noted that no significant temperature effect comparable to the ease of L-cystine was observed, as shown in Fig. 4. it is strongly suggested that tile population among conformers produced by internal rotations of the above-mentioned single bonds is not appreciably affected bv the change of temperature between - 7 °° and I8 °. in case of L-cystine, the situation where peripheral effects are concerned, seems not so different from the above cases. At the present time, the viewpoint that the peripheral effects dominate the CD band at 255 nm cannot be easily accepted. If the peripheral effects do not change appreciably with temperature, ttle observed change of CD of L-cystine solution (Fig. 3) can be ascribed to the change in the disulfide groups between M and P conformations. Even if Curve 2 in Fig. 5 was assumed chieflv to reflect the shift of equilibrium between P and M conformations, further analysis of the curve should be suspended, because the extrapolation was arbitrary, and no reliable estimate of the CD intensity of each conformer was possible. Biochim. 13iophys. dcta, 257 (I972) i-io

CIRCULAR DICHROISM OF CYSTINE

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Apart from the origin of the observed temperature dependence, the present results have a significant implication in the interpretation of CD spectra of proteins and peptides containing a cystine residue. Curve 2 in Fig. 5 should be extrapolated to the CD intensity ranging between -- 4400 and -- 5500 °. cm 2. dmole -1 at lower temperature. This indicates that the more stable conformer(s), that become predominant at a lower temperature, have a bigger CD intensity in the first and third CD bands than that expected from the CD spectrum measured at around 20 °. The small negative CD intensity attained at higher temperature also suggests that the less stable conformer(s), of which the population is expected to increase to the extent comparable to that of the more stable one(s), have a big positive CD intensity slightly smaller than Lllat of the latter in magnitude. Most cystine residues in a globular protein molecule are expected to have fixed conformations. As in the lysozyme molecule TM, they are expected to have either M or P conformation. The present results strongly suggest that a fixed cystine residue in a protein molecule can have a bigger CD intensity than that expected from the CD spectrum of L-cystine solution measured at around 20 °. The sign depends on the assumed conformation. Therefore, if cystine residues having the same sign of CD intensity are predominant in a protein molecule, cystine residues are expected to make a significant contribution to the observed CD spectrum at least in the near ultraviolet region. On the other hand, if conformations of cystine residues in a protein or peptide molecule are not well fixed, its CD spectrum is expected to change with temperature. Significant temperature dependence of CD in the near ultraviolet region has been reported with oxytocin and its derivatives 21, ribonuclease 22 and insulin 23. Seemingly due care should be taken in interpreting the observed results of the dependence of CD of L-cystine residue on temperature. We have observed that N,N'-diaeetyl-L-cystine bismethylamide, which more resembles the cystine residue in a polypeptide chain than L-cystine, shows similar temperature dependence in its CD spectrum to that observed with L-cvstine (T. TAKAGI, R. OKANO AND T. MIYAZAWA, unpublished results). As has been reported by COLEMAN AND BLOUT 13, the cystine derivative has a negative CD band at 26 7 nm.

ACKNOWLEDGEMENTS

We wish to express our appreciation to Prof. T. Isemura for his encouragement. We also thank Prof. T. Miyazawa for stimulating discussion. REFERENCES I 2 3 4 5 6 7 8 9 io ii

R. S, CAHN, C. INGOLD AND V. PRELOG, Angew. Chem. Int. Ed. Engl., 5 (1966) 385 . C. DJERASSI, Optical Rotatory Dispersion, M c G r a w - H i l l , New Y ork, 196o, p. 225. S. BEYCHOK, Science, 154 (1966) 1288. W. G. PENNEY AND G. B. B. M. SUTHERLAND, J. Chem. Phys., 2 (1934) 492. B. M. OUGHTON ANn P. M. HARRISON, Acta Crystallog., 12 (1959) 396. J. PETERSEN, L. K. STEINRAUF AND n. H. JENSEN, Acta Crystallog., 13 (196o) lO 4. A. F, CORSMIT, A. SCHUYFF AND D. FELL, Proc. K. Ned. ~1had. Wet. Ser. C., 59 (1956) 47 °. L. K. STEINRAUF, J. PETERSEN AND L. H. JENSEN, J. Am. Chem. Soc., 89 (1958) 3835 . H. L. YAKEL AND E. W. HUGHES, dcta Crystallog., 7 (1954) 291. C. C. BLAKE, G. A. MAIR, A. C. T. NORTH, D. C. PHILLIPS AND V. R. SARMA, Proc. Roy. SOC. Lond. Ser. B, 167 (1967) 365 . G. TOENNIES AND T. F. LAVINE, J. Biol. Chem., 89 (193 o) 153.

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I2 13 14 15 16 17 18

C. SZANTAY, M. P. IKOTICK, E. SHEFTER AND T. J. BARDOS, J. An.t. Chem. Soc., 89 (1967) 713 • D. L. COLEMAN AND E. R. ]~LOUT, J. A~n. Chem. Soc., 90 (1968) 2405. Y. NOZAKI AND C. TANFORD, J. Am. Chem. Soc., 89 (1967) 736L. F. FIESER, Rec. Tray. Chim. Pays-Bas, 69 (195o) 41o. L. VELLUZ AND M. LEGRAND, Angew. Chem. Int. Ed. Engl., 4 (1965) 838. J. LINDERBERG AND J. MICHL, J . Am. Chem. Soc., 92 (197 o) 2619. M. CALVI,',', in S. COLOWlCK, A. LAZAROW, E. RACKER, D. R. SCHWARTZ, F. STEDTMAN AND H. WAELSCH, Glutathione, Academic Press, New York, 1954, p. 3. G. BERGSON, Ark. Kern., i2 (1958) 233. G. BERGSON, Ark. Ix'era., 18 (1962) 409. D. Wr, URRY, F. QUADRIFOGLIO, t~. V~TALTER AND L. L. SCHWARTZ, Pt'OC. Natl, Acad. U.S., 60 (1968) 967. M. lx~. PFLUMN AND S. BEYCHOK, ./. Biol. Chem., 244 (1969) 3982. M. J. ]~TTINGER AND S. N. TIMASHEFF, Biochemistry, io (1971) 824.

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