Dye-sensitized selective photooxidation of methioxine

BIOCHEMICA ET BIOPHYSICA ACTA

B~A 35163 D Y E - S E N S I T I Z E D SELECTIVE P H O T O O X I D A T I O N OF M E T H I O N I N E

G I U L I O J O R I , G U I D O GALIAZZO, A R M A N D O M A R Z O T T O AND E R N E S T O S C O F F O N E The Islitulo di Chimica Organica dell' Universild di Padova and Cenlro Nazionale di Chirnica delle Macromolecole del C.N.R., Padova (Ilaly) (Received A u g u s t 9th, 1967)

SUMMARY

The photooxidation, sensitized by rose bengal and methylene blue, of cystine, methionine, histidine, tyrosine, tryptophan and of some related peptides was studied in acid media. Rose bengal in formic acid solution, and methylene blue or rose bengal in aqueous acetic acid solution sensitized a selective oxidation of methionine, which was quantitatively converted to methionine sulphoxide. Irradiation of ribonuclease A (ribonucleate pyrimidinenucleotide-2'-transferase (cyclizing), EC 2.7.7.16 ) under the same conditions caused the modification of the four methionyl residues and a concomitant 87% decrease of the enzymatic activity, which was correlated to a conformational change of the protein. Chemical reduction of the photooxidized ribonuclease A by thioglycolic acid resulted in the full recovery of the enzvmatic activity.

INTRODUCTION

Dye-sensitized photooxidations have been widely employed for the modification of the tryptophyl, tyrosyl, histidyl and methionyl residues in many proteins and enzymes. The reaction rate is strongly dependent on the pH of the reaction medium and on the sensitizer used1 : in any case, at pH >6, all four amino acid residues are oxidized at comparable rates~, a. This lack of specificity led us to investigate the sensitivity of amino acids and peptides to the photodynamic action of various dyes in acid media. In a previous paper 4, we proposed the irradiation by visible light in formic acid solutions and in the presence of proflavine as a method for the selective photooxidation of the tryptophyl residues. In this paper, we report our findings about the sensitizing action of rose bengal and methylene blue. These dyes were chosen because some photodynamic data about them had already been publishedS, 6 and they had been successfully used as sensitizers in the photooxidation of biological substrates 7 14 The irradiations were carried out on both the free amino acids and some related peptides, since it was shown that the incorporation into a polypeptide chain may affect the course of the oxidative breakdown of an amino acid moleculO. Moreover, comparison of the rate constants for the photooxidation of the susceptible amino acids, when free or bound in a protein, permits one to differentiate between the accessible and inaccessible residues 15. As a model, Biochim. t~iophys. Acta, 154 (1968) 1-9

2

G. JORI el a/.

the principal chromatographic component of pancreatic bovine ribonuclease, ribonuclease A (ribonucleate pyrimidinenucleotide-2'-transferase (cyclizing), EC 2.7.7.16), was chosen; its primary structure is well known, and it contains no tryptophan (i.e. a highly photoreactive substrate), therefore allowing an easier interpretation of the chemical and biological changes carried out. MATERIALS AND METHODS Materials The amino acids were Fluka products. Peptides N-carbobenzyloxy-fl-alanyl-I.histidine methylester (I), N-carbobenzyloxy-fl-alanyl-L-tyrosine methylester (II), Ncarbobenzyloxy-L-tryptophyl-glycine methylester (III), N-carbobenzyloxy-L-tryptophyl-L-methionine methylester (IV), N-carbobenzyloxy-fl-alanyl-L-tryptophylglycine ethylester (V), were synthetized in this institute. Peptide L-glutamyl--L-histidyl-L-phenylalanyl-L-arginyl-L-tryptophyl-glycine (VI) was a gift of Dr. W. RITTEL of CIBA (Basle, Switzerland). Pancreatic bovine ribonuclease A obtained from Worthington Biochemical and purified according to the procedure of HIRS, STEIN AND MOORE16, was used as a standard protein. The enzymic activity of the dye-freed, photooxidized ribonuclease A was checked by using yeast RNA (Schwartz Bioresearch) as substrate, according to the KUNITZ procedurO 7, as modified recently b y MARZOTTO et al) s. Rose bengal B (3',4',5',6'-tetrachloro-2,4,5,7 -tetraiodofluorescein disodium salt) and methylene blue were obtained from Fluka. Formic acid and acetic acid were obtained from Merck. Sephadex G-25 was the product of Pharmacia (Uppsala, Sweden), Amberlite CG-5o was obtained from British Drug Houses. All other reagents were commercial analytical grade products, unless otherwise stated. Irradiations The experimental procedure was the same as previously described4; samples were irradiated at a distance of 40 cm, in io ml Pyrex test tubes b y four I5o-W incandescent light bulbs, placed on either side of a transparent bath with plexiglass walls. The temperature was maintained at i ° or 37 ° by circulating water. In each experiment, 2 ml of a 2-raM substrate solution were added to 2 ml of a o.2-mM dye solution in the dark; when temperature equilibrium was reached, the lights were switched on and a stream of purified 02 was slowly fluxed through the solutions. Controls without light or dye were also run. The following reaction media were tested: p H 5 and p H 3 buffered solutions (0.2 M NaH2PO4), 0-50% aq. mixtures of formic acid and acetic acid. A m i n o acid analysis In the case of amino acids and oligopeptides, the reaction was followed b y chromatographic and spectrophotometric analyses. Chromatographic techniques involved both descending paper chromatography on W h a t m a n No. I, using the PARTRIDGE mixture 19 or 5 ~o ammonia as eluent, and ascending thin-layer chromatography on silica gel, using the solvent systems: (I) nbutanol-water-acetic acid (4 :I :I, v/v/v), (2) methylacetate-isopropanol-I7 % ammonia (2 : 2 :I, v/v/v), (3) chloroform-benzene-acetic acid (16:3 :I, v/v/v). Biochim. Biophys. Acta, I54 (1968) 1-9

SELECTIVE PHOTOOXIDATION OF METHIONINE

3

Spots were developed by ninhydrin (o.4% butanolic solution) and, in the case of tryptophan derivatives, also by Ehrlich's reagent, modified according to STAHL AND VAN URKa°; for peptides the Reindel-Hoppe reagent ~1 was also used. Quantitative determinations were obtained by automatic amino acid analysis, according to t h e m e t h o d o f SPACKMAN, STEIN" AND MOORE 22.

Spectrophotometric analyses were carried out by an Optica CF 4 DR and a Beckman DU spectrophotometer. Prior to analysis, the dye was removed from the solution with Norit, or by paper chromatographic separation of the irradiated mixture and subsequent elution of the product with water or 95% ethanol. For methionine, tryptophan and related peptides, the analytical method previously described 4 was used for quantitative determinations. In the case ofribonuclease A, the irradiation was carried out in 84% acetic acid. At the end, the solvent was removed by lyophilization, the residue was taken up with 5% acetic acid, and the solution was freed of the dye by running it over a column (1.2 c m x 58 cm) ofSephadex G-25, medium grade, using 5% acetic acid as the eluent. Samples for amino acid analysis were hydrolyzed in 6 M HC1 in evacuated sealed tubes at i i o ° for 22 h. Alkaline hydrolyses were also performed in order to evaluate the content in methionine sulphoxide : for this purpose, about 5 mg of tlle photooxidized protein were dissolved in 1.5 ml of 3.70 M NaOH in evacuated sealed quartz vials and heated at ioo ° for 16 h; then the samples were cooled, acidified to pH 1. 5 with 6 M HC1, and automatically analyzed.

Regeneration of native ribonuclease A In an experiment, 37 mg of photooxidized ribonuclease A were reduced by 1% aqueous thioglycolic acid, as described by HOFMANNet al. 23. The fully inactive product obtained (32 rag) was dissolved in a o.I M Tris solution (pH 8.1) at a concentration of 0.2 mg/ml, and exposed to air for 24 h at 23 °, according to the procedure described by HABER AND ANFINSEN24. The protein solution was desalted by gel filtration on Sephadex G-25, using 5 % acetic acid as eluent, and the solvent was removed by lyophilJzation. The yield was 26 rag. RESULTS

Photooxidation of amino acids and oligopeptides In all the reaction media studied, the ultraviolet spectra and the chromatographic pattern of the irradiated solutions of cystine, histidine and tyrosine, as well as of Peptides I and II, were coincident with the ones of the corresponding non-irradiated solutions, even after prolonged exposure to light. From amino acid analysis, the recovery of these substrates were quantitative in every case. These results are in agreement with previous findings4, 25 about the lack of reactivity of the afore-mentioned amino acids in acid solutions. Tryptophan, on the contrary, showed notable changes in reactivity, according t(, the solvent and the sensitizer used. In Table I, the percent recovery of tryptophan after irradiation of the free amino acid and related peptides at 37 ° in different reaction media is reported. In the presence of methylene blue, this amino acid is very slowly photooxidized except in acetic acid solutions. In the presence of rose bengal, it is odixized both in Biochim. Biophvs. Acta, I54 (I968) I--9

C. JOR1 el al.

4 TABLE I PERCENT

RECOVERY

OF TRYPTOPHAN

AFTER

IRRADIATION

IN DIFFERENT

REACTION

MEDIA

The buffer used was o.2 M N a t l 2 P O a. P e p t i d e I I l , N - c a r b o b e n z y l o x y - L - t r y p t o p h y l g l y c i n e m e t h y l e s t e r ; P e p t i d e IV, N - c a r b o b e n z y l o x y - L - t r y p t o p h y l - L - m e t h i o n i n e m e t h y l e s t e r ; P e p t i d e V, N - c a r b o b e n z y l o x y - f i - a l a n y l L - t r y p t o p h y l - g l y c i n e e t h y l e s t e r ; P e p t i d e VI, L - g l u t a m y l L-hist i d y l - L - p h e n y l a l a n y l - L - a r g i n y I - L - t r y p t o p h y l - g l y c i n e . R e c o v e r y w a s measured after 3 ° min irradiation a t 37 ° .

Dye

Solvent

Substrate l'rvptophau

Methylene blue

Rose bengal

Peptide I l i

Peptide

Peptide

IV

V

Buffer pJcI 5 97 .6 Buffer p H 3 99.0 5 0 % acetic acid 71.2 8 4 % acetic acid 57.3 9 9 % acetic acid 35.8 9 9 % formic acid l o l . 3

--8.5.3 -76.1 98.5

87. 5 78.0 74.2: 98.9

Buffer p H 5 Buffer p H 3 5 0 % acetic acid 8 4 % acetic acid 9 9 % acetic acid 5 ° /o/ o formic acid 9 9 /o/ o formic acid

--

88.9 -83.6

86.9 72.3 90. 3 91. 3 86. 7

99.0

lOl. 3

84 .2 70.3 75.9 7o.1 43.6 99.6 98.9

98.7 -

97.9 96.5 --8o. 4

Peptide V I

--90.6 __ 82.5 lOO.5

85.3

--

--92-7 86. 5

93.6 -90.2 98. t

buffered and in acetic acid solutions, but not in formic acid solutions. Whereas in buffered solutions the degradation rate is about the same for tryptophan, whether free or bound in a peptide chain, in acetic acid solutions the incorporation of tryptophan into a peptide molecule is concomitant with a drop in the reaction rate. Moreover, the reaction rate is affected by temperature : e.g., after I h irradiation of the free amino acid at 37 ° in 99% acetic acid solution and in the presence of methylene blue, less than IO % of tryptophan is present; under the same conditions, but at I °, 65 % of tryptophan is still unreacted. This different behaviour of tryptophan, as well as of the related peptides, is to be ascribed not only to a different photodynamic efficiency of the dyes employed in the above discussed reaction media, but also to a different path in the breakdown of the tryptophan molecule. Actually, our findings pointed out that a slight change in the irradiation conditions may markedly affect the nature and the number of the tryptophan degradation products. Work is in progress in order to shed further light on the photooxidation mechanism of this important substrate. Methionine, in the presence of methylene blue, appeared to be susceptible of photooxidation only in acetic acid solutions, where a very fast reaction takes place (see Table II) ; in the presence of rose bengal, this amino acid is photooxidized in all the reaction media studied. Whereas in buffered solutions the reaction rate is independent of pH, in acetic acid and in formic acid solutions it increases with increasing acid concentration. Temperature does not appreciably affect the reaction rate. Chromatographic analysis in different solvents of the irradiated methionine solutions showed that the amino acid was converted to one product, which was indistinguishable, according to RE values, from a sample of chemically prepared methionine sulphBiochim. Biopkys. Acta, t54 (lq681 1- 9

SELECTIVE PHOTOXIDATION OF METHIONINE

5

T A B L E II PERCENT

RECOVERY

OF METHIONINE

AFTER

IRRADIATION

IN DIFFERENT

REACTION

MEDIA

T h e buffer used w a s 0.2 M N a H 2 P O 4. P e p t i d e IV, N - c a r b o b e n z y l o x y - n - t r y p t o p h y l - L - m e t h i o nine m e t h y l e s t e r . R e c o v e r y was m e a s u r e d after 3 o - m i n irradiation at 37 °.

Dye

Solvent

Subsirale Nlethionine

3lelhy!ene blue

Rose bengal

Buffer p H 5 Buffer p H 3 5 0 % acetic acid 8 4 % acetic acid 9 9 % acetic acid 5 o % formic acid 9 9 % formic acid

97.2 95.0 6. 7 4.2 o.o 96.1 98. 3

Buffer p H 5 Buffer p H 3 5 0 % acetic acid 8 4 % acetic acid 9 9 /o/ o acetic acid 50 % formic acid 9 9 % formic acid

55.7 56.3 9.8 8.i 4-3 44.2 19.6

Peplide I V 95-I 96.5 7-9 3.6 1.8 IO1-3 56.1 58.8 8.8 5-7 23.6

oxide. From amino acid analysis, the yield of the conversion of methionine to its sulphoxide was found to be over 95 %, independent of dye, solvent and temperature. In order to compare the reactivity of the two susceptible amino acids; i.e. tryptophan and methionine, we studied the photooxidation kinetics of N-carbobenzyloxyL-tryptophyl-L-methionine methylester, under different conditions. The greatest differentiation between the two residues was found to occur on irradiation in aqueous acetic acid at i °.

100

_

80r

-e_

70L cJ

o

40 a0 20 10 0

~0

4'0

6'o

0'° IRRAOIATION

,;6 TIME

60 (MINUTES)

Fig. 1. P h o t o o x i d a t i o n rate o f the m e t h i o n y ] ( ~ Q ) and o f the t r y p t o p h y ] ( ~ - - - - O ) residue on i r r a d i a t i o n o f lO m M N - c a r b o b e n z y l o x y - L - t r y p t o p h y ] - L - m e t h i o n i n e m e t h y l e s t e r a t i ° in 8 4 % acetic acid so|ution and in the presence o f o.1 m M methylene blue.

Biochim. Biophys. Acla. 154 (1968) 1-9

G. JoRI el (¢1.

6

As shown in Fig. I, when all of methionine has disappeared, more than 9o~!.b of tryptophan is still unchanged. One may conclude that rose bengal in formic acid solution, and rose bengal or methylene blue in aqueous acetic acid solution sensitize a selective photooxidation of the methionyl residues in a polypeptide molecule. In every case, the control solutions without light or dye remained unchanged.

Photooxidation of ribonuclease A At first, we checked the stability of ribonuclease A in the reaction media, which were found to be specific for the photooxidation of methionine. After IO h storage in 98% formic acid solution or in 84~o acetic acid solution at I °, a fully active sample of ribonuclease A was recovered. No changes in the enzymatic activity were observed after io 11 irradiation of the protein at I °, in the same solvents and in the absence of dye. Therefore, the subsequent irradiations of ribonuclease A, in the presence of dye, were carried out in 84<7o acetic acid at I °, owing to the stability of the protein molecule under such conditions. Photooxidation of ribonuclease A, sensitized by methylene blue, was attended by a progressive loss of enzymatic activity, which was reduced to 48% after o.5 h, and to 13% after 2 h irradiation. Prolonging the exposure to light caused no further decrease in the enzymatic activity. Similar results were obtained if rose bengal was used as the sensitizer. As shown in Fig. 2, photooxidation of ribonuclease A caused a shift of the absorption maximum from 277. 5 m# to 276 m/t; this effect has been observed for several other reactions, all of which involve the denaturation of the molecule~6,2L

10

70

i6~

0 250

9 zz ~

270 290 310 WAVELENGTH (m~)

Fig. 2. Ultraviolet spectra of native ( - -

- - ) and of photooxidized ribonuclease A (

----).

Amino acid analyses of the samples of ribonuclease A inactivated to the extent of 87% are given in Table III. The values obtained after acid hydrolysis were in good agreement with the ones obtained from the controls : the slight difference in the methionine content was clearly balanced by the presence of traces of methionine sulphoxide. The measured number for tyrosine was appreciably low both in the photooxidized sample and in the control: at least part of this low recovery is caused by the formation of chlorotyrosine during acid hydrolysis 28. These data suggest that no amino acid residue was modified on photooxidation ; this conclusion, however, does not take into account a possible feverBiochim. Biophys. Acta, 154 (1968) I 9

SELECTIVE PHOTOOXIDATION OF METHIONINE T A B L E lI'~ AMINO

ACID

ANALYSES

OF RIBONUCLEASE

Amino acid

;3x

Residues per molec:de

Methionine sulphoxide Aspartic acid Threonine Serine G l u t a m i c acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine

sion of methionine

Theory

Native, acid hydrolysis

Photooxidized, acid hydroid'sis

o 15 lO 15 12 4 3

o i4. 4 9.6 14.1 I I. 8 3.9 3.3

Traces 14.6 io.3 14. 4 I 1.6 3.7 2-7

12

I2,2

I 1.8

8 9 4 3 2 6 3 io 4 4

7.7 8.5 3 .8 2.5 2.2 5.3 2.7 9- 6 3.8 3.7

sulphoxide

7.4 8.5 3. i 2.7 2. 4 5. I 2.9 i o.o 3.8 3 .8

to methionine

during the acid hydrolys is , as pointe d

o u t b y R A Y AND KOSHLAND 29. A c t u a l l y , b y a l k a l i n e h y d r o l y s i s , 3-7 r e s i d u e s o f m e thionine sulphoxide and no methionine

were found to be present. Therefore

on the

l '2

6

7

e,

9

10

17

12

13

pH

Fig. 3. Tyrosine t i t r a t i o n curves of native ( O - - © ) and of photooxidized ribonuclease A ( [ ] - - []). The molar extinction at 295 m/* is given as a function of p H . The different p H values were obtained using a o.i-M piperidine buffer adjusted to the desired value by the addition of s t a n d a r d HC1 or N a O H .

Biochim. Biophys. Acta, 154 (1968) I - 9

8

G. JOR1 et al.

basis of the amino acid analysis, the conclusion may be drawn that oxidation of the methionine sulphur is the sole modification which has been brought about. In order to check whether the observed decrease in the enzymatic activity of the oxidized ribonuelease A could be correlated with a conformational change of tile protein molecule, a sample of irradiated ribonuclease A was subjected to the spectrophotometric titration for tyrosines, according to the procedure described by RICHARDS AND LOGUE 3°.

As shown in Fig. 3, at least 4 tyrosyl residues were exposed in the modified protein. Accordingly, the large inactivation of the photooxidized ribonuclease A must be ascribed to the involvement of the methionyl residues in a conformational change which would pull at least one tyrosyl residue out of the hydrophobic environment in the interior of the molecule.

Regeneration of native ribonuclease A Since methionine sulphoxide is easily reduced to inethionine by the use of sulphydryl compounds, we tested whether the enzymatic activity of the oxidezed ribonuclease A could be recovered by chemical reduction, as described in tile experimental section. The completeness of the reaction was checked by amino acid anMysis after alkaline hydrolysis. The end-product, which contained no sulphoxides, was 98 ~;, as active as the native protein. Moreover, its ultraviolet spectrum and its chromatographic pattern on Amberlite CG-5o (o.9 cm × 60 em), using 0.2 M phosphate buffer pH 6.47 as eluent, were coincident with the ones of the native enzyme. These results suggest that, on the reversion of methionine sulphoxide to methionine, a regeneration o*-the native secondary and tertiary structures had occurred. DISCUSSION

Two features of the described photooxidation method deserve special attention. First, owing to the large sensitivity of tryptophan to temperature and to the nature of dye and solvent, the photodynamic action of methylene blue and rose bengal can be made to act selectively on methionine by irradiation in formic acid solution or in aqueous acetic acid solution at I °. Second, the end-product of the reaction is methionine sulphoxide, which easily reverts to methionine by reduction with sulphydryl compounds. Therefore, a new way is open for the specific and reversible modification of the methionyl residues in a polypeptide chain ; this result is difficult to obtain bv a chemical approach, owing to a scarcity of suitable reagents. The usefulness of the method, when applied to proteins, is apparent from the data obtained in the photooxidation of ribonuclease A. The possibility of operating at low temperatures protects the protein molecule from irreversible denaturation in the acid media employed, as was found by KOCH, LAMONTAND KATZal and confirmed by us. Moreover, the enzymatic activity, which had been extensively lowered by the photooxidation of the methionyl residues, is fully restored by treatment with I °/o thioglycolic acid. As indicated also by the spectrophotometric and chromatographic analyses, the reversibility of the method is not limited to the oxidative modification carried out on the methionyl residues, but includes the refolding of the protein molecule into the native conformation. Photooxidation of ribonuclease A in alkaline buffered solutions had been pre-

Biochim. t3iophys..4cta. 154 (1968) 1-9

S E L E C T I V E P H O T O O X I D A T I O N OF M E T H I O N I N E

9

viously performed by WEIL AND SEIBLESa2 and by KENKARE AND RICHARDS14. In both cases, photooxidation was attended by a loss of enzymatic activity, which was correlated by the authors to a decrease in the histidine content of the proteins. No methionine was found to be affected during the reaction. The discrepancy between these results and ours is due to the analytical method followed by other authors for the estimation of the amino acid residues in the photooxidized protein : i.e., acid hydrolysis az or performic acid oxidation prior to acid hydrolysis '4. Under these conditions, methionine sulphoxide is known to be reduced to methionine or oxidized to the sulphone, respectively. On the contrary, our findings point out that the photooxidation of the methionyl residues alone causes an 87 % inactivation of the protein. NEUM~NN, MOORE AND STEIN 33, after reaction of ribonuclease A with H202, isolated an active monosulphoxide derivative; however, samples in which more than one methionyl residue had been oxidized or carboxymethylated with iodoacetic acid were inactive. In our opinion, therefore, it is not possible to draw any definite conclusion about the role performed by an amino acid residue in a biologically active polypeptide from a photooxidative investigation, unless a modification of methionine has not been prevented or the sulphoxide derivatives formed are chemically reduced as indicated above. REFERENCES I A. D. MCLAREN AND D. SHUGAR, in P. ALEXANDER AND Z. M. BACQ, Photochemistry of Proteins and Nucleic Acids, Pergamon Press, London, 1964, p. 156. 2 L. WELL, W . G. GORDON AND A. R. BUCHERT, Arch. Biochem. Biophys., 33 ( I 9 5 I ) 90. 3 L. A. 3t2. SLUYTERMAN,Biochim. Biophys. Acta, 6o (I962) 557. 4 C. A. BENASSI, E. SCOFFONE, G. GALIAZZO AND G. JORI, Photochem. Photobiol., 6 (1967) 857. 5 G. OSTER AND A. H. ADELMANN, J. Am. Chem. Soc., 78 (1956) 3977. 6 G. OSTER, J. S. BELLIN, R. XV. KIMBALL AND M. E. SCHRADER, J. Am. Chem. Soc., 81 (1959) .5095. 7 G. RODEGHIERO AND C. BERGAMASCO, Farmaco (Pavia) Ed. Sci., 13 (1958) 368. 8 J. S. BELLIN AND G. OSTER, Biochim. Biophys. Acta, 42 (196o) 533. 9 K. SONE, Nippon Nogeikagaku Kaishi, 36 (1962) 7. l o L. X,VEIL AND T. S. SEIBLES, Arch. Biochem. Biophys., 54 (1955) 368. 11 J. \\:. 1RAV AND D. E. KOSHLAND ,J. Biol. Chem., 237 (1962) 2493. 12 G. \VEITZEL, W. SCHAEG, G. BONEY AND B. WlLLMS, Ann. Chem., 689 (1965) 248. ~3 B. R. DAS GUPTA ANn D. A. BOROFF, Biochirn. Biophys. Acta, I i O (I965) 57. 14 U. W . KENKA~eF. AND F. M. RICHARDS, J. Biol. Chem., 241 (1966) 3197 . I 5 \V. J. ]~AY AND 1). E. NOSttLAND, J. Biol. Chem., 236 (1961) 1973. I 0 C. \V. H. HIRS, \V. H. STEIN AND S. MOORE, J. Biol. Chem., 200 (1953) 493. 17 M. K u N I r Z , J. Biol. Chem., 164 (1946) 563 . 18 A. MARZOTTO, F. MARCHIORI, L. MORODER, i{. BONI AND 1,. GALZIGNA, Biochim. Biophys. Acta, 147 (1967) 26. 19 S. M. PARTRIDGE, Nature, 158 (1946) 270. 20 E. STAHL, Diinnschicht-Chromatographie, Springer Verlag, Berlin, 1962, p. 503 . 21 H. M. RYDON AND P. \V. S. SMITH, Nature, 169 (19.52) 922. 22 D. H. SPACKMAN, ~V. H. STEIN AND S. MOORE, Anal. Chem., 3 ° (1958) 119o. 23 K. HOFMANN, F. M. FINN, M. LIMETTI, J. MONTIBELLER AND G. ZANETTI, J. Am. Chem. Soc., 88 (1966) 3633 . 24 E. HAr3ER AND C. B. ANFINSEN, J. Biol. Chem., 237 (1962) 1839. 25 L. ~VE1L, Arch. Biochem. Biophvs., I i O (1965) 57. 26 C. B. ANEINSEN, J. Biol. Chem., 221 (1956 ) 405 . 27 F. H. WHITE, J. Biol. Chem., 236 (1961) 1353. 28 C. W . H. HIRS, J. Biol. Chem., 219 (1956 ) 611. 29 W . J. RAY AND D. E. KOSHLAND, Broohhaven Syrup. Biol., 13 (196o) 135. 3 ° F. M. I~ICHARDS AND A. D. LOGUE, J. Biol. Chem., 237 (1962) 3693 . 31 A. L. KOCH, W ~. A. LAMONT AND J. j . KATZ, Arch. Biochem. Biophys., 63 (1956) lO6. 32 L. \VEIL AND T. S. SEIBLES, Arch. Biochem. Biophys., 54 (1955) 368. 33 N. P. ~N-EUMANN, S. MOORE AND "vV. H. STEIN, Biochemistry, I (1962) 68.

Biochim. Biophys. Acla, 154 (1968) 1 - 9