BIOCH]~MICA ET BIOPHYSICA ACTA 345 BBA 26168 THE PHOTOCHEMICAL DEGRADATION OF CYSTINE IN AQUEOUS SOLUTION IN THE PRESENCE OF AIR R. S. ASQUITH A~D L. HIRST School of Colour Chemistry, University of Bradford, Bradford 7, Yorkshire (Great Britain) (Received March 6th, 1969) SUMMARY i. Ultraviolet irradiation of cystine in air-saturated aqueous solution at pHi and io has been investigated. 2. The quantum yield for destruction of cystine has been shown to be inde- pendent of pH, the increase in destruction rate at pH IO being explained by the increased absorption of energy at this pH. The reaction shows first-order character- istics when the intensity of the incident is in excess of that absorbed by cystine. 3-Degradation of cystine occurs only when the wavelength of the incident radiation is below 30o nm. 4-A large number of products have been confirmed, notably pyruvic acid, cysteine, ammonia, cysteic acid, alanine 3-sulphinic acid, cysteine S-sulphonic acid, alanine, serine, glycine, SO42- and, in acid solution, lanthionine, bis-(2-amino-2-car- boxyethyl)trisulphide, bis-(2-amino-2-carboxyethyl)tetrasulphide and H2S. NH2OH has been identified at both pH values. 5. It has been deduced that the difference in products formed in acid and alkali is due to secondary reactions rather than to any difference in the original fission of the cystine links. 6. Evidence suggests that fission of C--S, S-S and C-N links occur in acid and alkaline solution. 7. No decarboxylation products of cystine have been detected. 8. A reaction mechanism has been postulated which accounts for the products identified and for the amounts formed. INTRODUCTION Ultraviolet irradiation of proteins 1 causes degradation of several amino acids including cystine2, 3. It has been suggested that the inactivation of enzymes 4, 5 and the yellowing of keratin fibers ~-9 may be due to this decomposition. The earlier work on cystine degradation has been well reviewed by SAVIGE AND MACLAREN 1°. In some of this work experimental detail is lacking, and the products formed were difficult to identify without modern chromatographic techniques. MORI 1~, using paper chro- matography, was able to identify many of the simpler products from the irradiation of cystine solutions by a mercury vapor lamp. In all of this earlier work no products containing the intact S-S bond were reported. Biochim. Biophys. Acta, 184 (1969) 345-357
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`340 R.S. ASQUITH, L/HIRST SAVIGE and coworkers 12-15 have studied the photochemical degradation of cystine in greater detail. By using several ultraviolet sources, they obtained data which they tried to correlate with such parameters as pH of solution, concentration of cystine and the predominent wavelength of incident radiation. Many products were reported, some of which were only formed in trace amounts and some of which contained the intact S-S bond. Recently pyruvic acid formation has been suggested as arising via ~-amino-o~'-oxo-fl,fi'-dithiodipropionic acid during irradiation of cystine solutions 1~. The present work presents quantitative data on the production of the major products of which many have been previously qualitatively reported. EXPERIMENTAL Materials The following commercially available chemicals used were L-cystine, pyruvic acid (Hopkin and Williams), L-cysteine. HC1, L-cysteic acid, DL-alanine, DL-serine, glycine, taurine, NH2OH (B.D.H.), L-thiazolidene-4-carboxylic acid (K and K Labs.) and DL-lanthionine (Nutritional Biochemicals Corp.). Where necessary the amino acids were recrystallized. DL-Diaminoadipic acid was prepared by the method of SHEEHAN AND BOLHOFFER12; alanine 3-sulphinic acid by the method of EMILIOZZI AND PICHATlS; cysteine S-sulphonic acid by the method of $6RB019; and bis-(2-amino- 2-carboxyethyl)trisulphide by the method of FLETCHER AND ROBSO~ 2°. Bis-(2-amino- 2-carboxyethyl)tetrasulphide was prepared in solution by allowing the trisulphide to stand in dilute HC1. Automatic amino acid analysis of this solution showed two extra peaks of which one belonged to cystine and the other presumably was due to the tetrasulphide formed according to the equation2°: 2 Cys-S-S-S-Cys --+ Cys-S-S-Cys + Cys-S-S-S-S-Cys. The impure adduct of pyruvic acid and cysteine was prepared by the method of SCHUBERT 21. On heating in aqueous solution at IOO °, this compound gives impure thiazolidene-2-methyl-2,4-dicarboxylic acid 22. The presence of the latter was con- firmed by the synthesis of RIVETT et al. 23. While the structure of the former compound is unknown, the simplicity of its formation suggests that it may be the mercaptal CH 3 I Cys-S-C-S-Cys I COOH Ultraviolet spectra of the cystine solutions The absorption spectra were determined on a Hilger Watts Uvispec H7oo. 0.40 g cystine was dissolved in I 1 of distilled water containing either IO ml of concen- trated HC1 or io ml of 0.880 M ammonia. The absorbance of these solutions was plotted against wavelength with the solvent as blank. Preparation of the cystine solutions Acid solutions of cystine were prepared by dissolving I.O g cystine in IOO ml distilled water containing io ml A.R. 35 % HC1 and by diluting it to I 1 with distilled Biochim. Biophys. Mcta, 184 (1969) 345-357
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`THE PHOTOLYSIS OF CYSTINE 347 water. Alkaline solutions were made by dissolving I.O g cystine in 5oo ml distilled water containing IO ml A.R. concentrated ammonia solution and by diluting it to I 1 with distilled water. All solutions contained 4.15 #moles cystine/ml. Irradiation of cystine solutions All the solutions were irradiated with a Hanau type Q8I high pressure mercury vapor immersion lamp protected by a quartz sheath. The solutions were circulated as a 7-ram layer around the lamp at a constant rate with a peristaltic pump. The temperature was maintained at 15 °. Samples were withdrawn for analysis as required from the open reservoir included in the cycle. I 1 of solution was used for each irradi- ation and all samples for amino acid analysis were immediately freeze-dried on with- drawal in order to reduce the possibility of subsequent reaction. Analysis of irradiated solutions High voltage electrophoresis was carried out on I5-cm wide Whatman 3 MM paper for 12o min between I m plates with a potential gradient of ioo V/cm at 5 °. The buffer (pH 1.85) consisted of 25 g formic acid and 78 g acetic acid diluted with distilled water to I 1. The residue from a 2-ml freeze-dried sample was diluted to 0.2 ml with distilled water; 5o-~1 aliquots were applied to the paper. After electro- phoresis was carried out, the paper was dried at 80 ° and was developed with cadmium acetate-ninhydrin reagent 24. The major bands were identified by direct comparison with controls of known materials. Subsequent chromatography in the second di- mension with n-butanol-glacial acetic acid-water (4:1:1, by vol.) showed that electrophoresis had resolved the bands into single substances. Quantitative amino acid analyses were carried out on a Technicon autoanalyser using the single column method of HAMILTON25with norleucine as an internal standard. Pyruvic acid was estimated colorimetrically by the method of FRIEDMANN and HAUGEN 26. Samples were analysed immediately without being freeze-dried. Ammonia was estimated colorimetrically with Nessler's reagent ~7. Analysis was carried out immediately without freeze-drying. Cysteic acid was estimated by electrophoresis at pH 1.85 (ref. 28). To detect NH20H after a io-h irradiation, a ioo-ml sample was taken to dryness in a rotary evaporator at 20 °. NH2OH was identified in this sample as the hydroxamic acid 29. This test was not sensitive enough to follow its formation quantitatively. To test for peroxides to IO ml of the acidified irradiated solutions 3 ml amyl- alcohol were added, and the mixture was shaken with three drops of KzCr20 7 solution. In the presence of peroxide a blue color should develop in the organic layer. No color formed in the irradiated samples, while controls to which traces of H~O 2 were added developed immediate color. RESULTS AND DISCUSSION On exposure of cystine to ultraviolet radiation from the immersed high pressure lamp, destruction of cystine commenced immediately and was virtually complete after a Io-h exposure. A more intense yellow color developed when cystine was ex- posed at pH io. This difference has been previously noted 3°, but the yellow pigment Biochim. Biophys. Acta, 184 (I969) 345-357
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`348 R.S. ASQUITH, L. HIRST has not yet been identified. Despite this observed difference in the rate of yellowing, the rate of decomposition of cystine on exposure to ultraviolet radiation is not so dependent on pH. The initial decomposition of cystine shows first-order kinetics with respect to the cystine concentration under the experimental conditions used, i.e., intensity of radiation in excess of that absorbed by the cystine, and a plot of log concentration of cystine against time is linear at both pH values. The color is therefore probably developed through secondary pH-dependent reactions. The rate is slightly greater at pH io (K ---- 6.54" lO -3) than at pHI (K ---- 5.50" lO-3). The quanta actually absorbed by the cystine during the irradiations have been calculated for the relevant line outputs of the lamp (Tables I and II); from these values the total number of TABLE I CALCULATED QUANTA OF ENERGY ABSORBED BY THE IRRADIATED ACIDIC CYSTINE SOLUTIONS AT VARIOUS ~rAVELENGTHS OF LIGHT (INITIAL VALUE) Wavelength Quanta/see A bsorbance Transmission Absorption Quanta/see of line from lamp * of solution* * of solution of solution absorbed by (nm) (x io 18) (%) (%) solution (x ±o 18) 248 0.324 0.900 13.o 87.0 0.282 254 1.59 o.825 15.o 85.o 1.35 265 0.688 0.577 26. 5 73.3 0.507 270 0.074 0.463 34.5 65.5 0.049 280 0.305 0.263 54.7 45.3 o.138 289 o.276 o.14o 72.5 27.5 0.076 297 o.693 0.070 85.0 I5.O o. lO 3 302 1.24 0.040 91.o 9.0 o.III 313 2.64 o.o17 96.o 4.0 o.lo 5 Total 2.721 * Supplied by the manufacturers. ** Corrected for path length and concentration. TABLE II CALCULATED QUANTA OF ENERGY ABSORBED BY IRRADIATED ALKALINE CYSTINE SOLUTIONS AT VARIOUS WAVELENGTHS OF LIGHT (INITIAL VALUE) Wavelength Quanta/sec A bsorbance Transmission Absorption Quanta/sec of line from lamp * of solution* * of solution of solution absorbed by (nm) (× /-018) (%) (%) solution (X I0 ls) 248 0.324 1.03 9.3 90-7 0.293 254 1.59 I.OO IO.O 90.0 1.43 265 O.688 o.850 15.7 84.3 o.582 270 0.074 0.675 21.3 78.7 0.058 280 o.3o 5 0.428 37.0 63.0 o. 19o 289 0.276 o.228 59.o 41.o o.113 297 0.693 o.131 74.0 26.o o.18o 302 1.24 0.086 82.o 18.o o.222 313 2.64 0.025 95.0 5.0 o.132 Total 3.2oo * Supplied by the manufacturers. ** Corrected for path length and concentration. Biochim. Biophys. Acts, 184 (1969) 345-357
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`THE PHOTOLYSIS OF CYSTINE 349 TABLE III THE INITIAL QUANTUM EFFICIENCY OF THE DECOMPOSITION OF CYSTINE IN ACIDIC AND ALKALINE SOLUTION Solutions initially contained 4.15/*moles/ml. pH of Percentage of Number of Number of 0"* solution total cystine moles molecules decomposed decomposed decomposed/ sec* I 58 0.00242 4.03. lO 17 o.15 IO 68 o.oo283 4-73" l°17 °.15 * An average value over the first hour. ** # is quantum efficiency, i.e., number of molecules decomposed/sec per number of quanta absorbed/see. quanta initially absorbed per sec by the cystine is found to be 2.72" IO is in acid and 3.20" lO 18 in alkali. This 18 % increase in absorption at the higher pH explains the 19 % increase in the decomposition rate of cystine at this pH. From the concentration curves (Fig. I), the number of molecules destroyed per sec (average value over the Ist hour) was found to be 4.03. IO ~ in acid and 4-73" IO~ in alkali (Table III). The quantum efficiency of the initial reaction was found to be independent on pH and to have a value of o.15 which is of the same order as the previously quoted value of o.13 (ref. 32) found in acid solution under N~. This suggests that the rate of the initial fission is independent of the O~ concentration. This behavior is consistent with a free radical mechanism for the initial breakdown of the cystine molecule. Previous authors using different techniques, e.g. polymerization initiation 31, electron spin reso- nance measurements ~*, disulphide interchangO 5, etc., on irradiation of disulphide • q 1.4 1.2 0.8 ~0.6 } o.4 8~ o.2 - 0 "0'20 1 2 3 4 5 6 7 8 9 10 Time of irrodiation (h) t 0.7 8 4--' .~0.6 o.~ ~ o.4 O ~ 0,3 L ~o.2 < 0.1 ~ .,1 i// , it/I \\/ 240 260 280 300 2~0 X(nm) I0O 90 BO 7o ~ ~,0 .-~ 50 .-- 4o ~ 3O ~ 20 ~ I0 0 Fig. I. The rate of decomposition of cystine plotted as log cystine concn, against time. Concn. expressed as/,moles of cystine remaining per/,mole of cystine originally present. Q--Q, at pHi ; A--~k, at pH IO. Fig. 2. The ultraviolet absorption spectra of cystine solutions showing the influence of pH on the absorption maximum. O--O, at pH I ; A--A, at pH io; ..... , transmission of the pyrex glass filter. Biochim. Biophys. Acta, 184 (1969) 345-357
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`350 R. S. ASQUITH, L. HIRST compounds suggest a free radical decomposition. Further irradiation in the presence of hydroquinone inhibits the formation of the normal breakdown products, none of which could be detected on electrophoresis after I h of irradiation. The cystine solution was irradiated using a pyrex glass filter instead of the quartz sheath; the transmission of the filter is shown in Fig. 2. The light source has strong lines emitted at 3o2 and 313 nm (Tables I and II), and although much of this radiation will pass through the filter, energy from lines below this will not. The wavelength of maximum absorption of cystine is 248 nm and is independent of the pH of the solution. Although some slight absorption occurs at 302 and 313 nm (Fig. 2), no detectable decomposition of cystine occurs at either pH after a 5-h irradi- ation with the filter. This indicates that light above 300 nm has little or no effect on cystine in a reasonable period of time. Thus all the sources of ultraviolet radiation used in previously reported work must have contained some radiation below 300 nm, even though some are stated to have maximum emission at a longer wavelength. Alternatively other workers may have used much higher intensities above 300 nm. In the present studies, after a 2-h irradiation, as many as twenty discrete nin- hydrin-positive bands were observed on electrophoresis of the irradiated solution at pH 1.85 (Fig. 3). Many of these products are, however, only present in trace amounts. Most of the major stable products have been identified by comparison with authentic samples on electrophoresis. Automatic amino acid analysis has been used for confir- [ I ~I ~' ~ < A J J pH 1 pH i0 II J I I [ll J 1flU Ill + ~ ~ m ~ ~, rJ ~) II I ] ] I] Bill ] B I [IpH11 I I]11 Fig. 3. A. Automatic amino acid analysis patterns showing the identified amino acids and major unknowns formed on photolysis of cystine at pHi and lo, respectively. B. Electrophoresis patterns showing the differences between the products formed on photolysis of cystine at pH i and io. The figures indicate the RF values of unknown products relative to glycine RF value IOO. Biochim. Biophys. Acta, 184 (1969) 345-357
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`THE PHOTOLYSIS OF CYSTINE 351 mation of their identity (Fig. 3) and also to plot their concentration as a function of time. This has been done for the following products: cysteine, lanthionine, serine, alanine, glycine, bis-(2-amino-2-carboxyethyl)trisulphide and tetrasulphide of which the standard equivalent was taken to be the same as the trisulphide. The formation of four other unidentified peaks ill acid solution was followed assuming their standard equivalent on the analyser to be i.o. Thiazolidene 4-carboxylic acid tentatively re- ported by FORBES AND SAVIGE la did not prove to be one of these unknowns, while the presence of ammonia and of cysteic acid was confirmed. Cysteic acid was not estimated on the analyser because several minor products with pK values of less than 2.8 7 (the pH of the initial eluent) were eluted from the column along with the cysteic acid. Ammonia was estimated directly in the irradiated solution to avoid loss during freeze-drying. Some of these products may be produced via less stable inter- mediates which are present only in trace amounts at any one time because of their rapid conversion to more stable products. Before considering these products it should be noted that while the overall decay of cystine is not affected by differences in pH of the exposed solution, the amounts and types of products are pH-dependent. The most noticeable difference between products from acid and alkaline solution is the complete absence in alkaline solution of lanthionine, bis-(2-amino-2-carboxyethyl)- trisulphide, bis-(2-amino-2-carboxyethyl)tetrasulphide and the three major unknowns formed in acid solution. Differences also occur in the amounts of other products formed at any given irradiation time. Taurine, mercaptoethylamine and the mixed disulphide Cys-S-S-CH~CH2NH 2 were not found at either pH value; this supports FORBES AND SAVIGE lz who found no evidence for decarboxylation. Sulphur-free amino acid products Fig. 4 shows the variation with time of the concentration of the sulphur-free amino acids produced in the irradiated solution. It can be seen, in agreement with previous suggestions, that glycine is only produced in very small amounts in acid and alkaline solution which shows that little side chain C-C bond fission occurs. Serine and alanine are formed in greater amounts than glycine at both pH values. Alanine 0.09 2.~ °o.o4 / ./~" I ~o.o2 , ~" • . ~ ~~o.~ . ..... 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 Time of irrocliation (h) Time of irrodicltion (h) Fig. 4. The concentrations of sulphur free amino acids produced on photolysis of cystine in acid and alkali plotted against time of exposure. •-'-0, glycine at pH I ; A-'-A, glycine at pH io; • --0, serine at pH i; A--A, serine at pH io; • ..... •, alanine at pH 1; A------A, alanine at pH IO. Fig. 5. The concentrations of amino acids containing one sulphur atom produced on the photolysis of cystine in acid and alkali plotted against time of exposure of the cystine. 0--0, cysteine at pHI ; A--~, cysteine at pH IO; @---@, cysteic acid at pHi ; A---A, cysteic acid at pH io; 0-'-0, lanthionine at pH i. Biochim. Biophys. Acta, 184 (1969) 345-357
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`352 R.S. ASQUITH, L. HIRST is produced in greater amounts than serine in acid solution and more rapidly in alkaline solution. Amino acid products containing one sulphur atom Fig. 5 shows the variation with time of the concentration of some sulphur- containing amino acids produced in the irradiated solution. Cysteine is produced at both pH values, but a greater amount is formed in acid. In both cases the concen- tration of cysteine fell to almost zero after a io-h irradiation. Cysteic acid is formed in greater amounts in acid than in alkaline solution, alanine 3-sulphinic acid is present in small amounts in both acid and alkaline solution, and lanthionine which is produced only in acid solution never reaches a concentration as high as cysteine. Lanthionine continues to decompose after reaching a maximum concentration. ~ aolc ~-F~ aoo~ ~ 000~ E ' *~ o.oo, ~ ~ o.oo~, 1 2 3 4 5 6 7 8 9 10 Time of irradiation (h) Fig. 6. The concentrations of amino acids containing the intact disulphide bond produced on photolysis of cystine in acid plotted against time of exposure of the cystine. I--I, bis-(2-amino- 2-carboxyethyl)trisulphide at pH i; 0--0, bis-(2-amino-2-carboxyethyl)tetrasulphide at pH i. Amino acid products containing the intact disulphide bond Fig. 6 shows the variation with time of products containing the intact S-S bonds formed in the irradiated solution. It can be seen that they increase to a maximum during irradiation before disappearing. This undoubtedly indicates that these products decompose under irradiation. Cysteine S-sulphonic acid was formed in small amounts at both pH values but was not quantitatively estimated; when irradiation was pro- longed this product was totally degraded. ~,. °- O. 4 ~ L~o.3 "; 0.1 o~ ~ 3 4 ~ 6 7 8 9 10 Time of irradiation (h) = ~0.07 k:_~ " go.ce 0.04 ~ 0.03 ~o.ce O.01 0 / / 1 2 3 4 5 6 7 8 9 10 Time of irr~dlotion (h) Fig. 7- The concentrations of the estimated non-amino acids formed on the photolysis of cystine in acid and alkali plotted against time of exposure of the cystine. Q--O, pyruvic acid at pHi ; Ak--(cid:127), pyruvic acid at pH io; II--I1, ammonia at pH i. Fig. 8. The concentrations of unidentified amino acids formed on photolysis of cystine in acid and alkali plotted against time of exposure of cystine. Biochim. Biophys. Acta, 184 (1969) 345-357
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`THE PHOTOLYSIS OF CYSTINE 353 Non-amino acid products Fig. 7 shows the variation with time of the non-amino acid products estimated. Pyruvic acid was formed at both pH values. It was only possible to estimate ammonia at pH i, since ammonia was used in the solvent to produce the higher pH. Investi- gation after irradiation of the final liquors for compounds other than amino acids also indicated the presence of inorganic sulphate, NH20H and, in acid, H2S. Major unidentified products Fig. 8 shows the changes in concentration of the unknown amino acids formed on irradiation. While product A is formed in acidic and alkaline solutions, Products B, C and D are only formed in acidic solution. Although mechanisms for the photolytic decomposition of cystine (excluding deamination) can be postulated involving initial homolytic fission of either the S-S or the C-S bonds in the molecule a3, neither of these mechanisms alone explains our results. Initial fission of only the S-S bond and two subsequent radical abstractions could be used to explain the presence of all the products formed, i.e., hv Cys-S-S-Cys -->2 Cys-S. k ~ -~r Cys-S-S-S-Cys + Cys" Cys-S' + Cys-S-S-Cys // (z) ''-~ Cys-S-Cys + Cys-S-S" Reaction I involves the production of bis-(2-amino-2-carboxyethyl)trisulphide and Cys. radicals in equimolar amounts. In acid solution the amount of the trisulphide is very small compared with the amount of alanine and serine derived from the Cys. radical. In alkali, alanine and serine but not the trisulphide are formed. Reaction 2 involves production of lanthionine and Cys-S-S. radical in equimolar amounts. In alkali, cysteine S-sulphonic acid derived from the Cys-S-S- radicals but not lanthionine is formed. This lack of correlation between the amounts of products formed indicates that fission of only the S-S bond in irradiated solutions of cystine is not sufficient to explain quantitatively the results obtained. A mechanism involving fission of only the C-S bond and subsequent radical abstraction using similar argu- ments is also unreasonable, i.e., hv Cys-S-S-Cys (3) Cys' + Cys-S-S-Cys (41 Cys-S-S + Cys-S-S-Cys > Cys-S-S" + Cys' > Cys-S-Cys + Cys-S. > Cys-S-S-S~Cys + Cys-S. Reaction 3 would result in equimolar amounts of lanthionine and Cys-S. radical being formed. Reaction 4 would result in equimolar amounts of bis-(2-amino-2-carboxy- ethyl)trisulphide and Cys-S. radical being formed. The small amount of lanthionine and trisulphide compared to the products derived from the Cys-S. radical in acid and the absence of lanthionine and the trisulphide in the presence of products derived from the Cys-S- radical in alkali rule out this mechanism at both pH values. Biochim. Biophys. Acta, 184 (1969) 345-357
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`354 R.s. ASQUITH, L. HIRST It is necessary therefore to use a mechanism involving simultaneous fission of the C-S bond in some molecules and the S-S bond in others to explain the results, i.e., k~ i~ r 2 Cys-S" / Cys-S-S-Cys / 76) ''~-''~ Cys-S-S" + Cys" The bond dissociation energy of C-S and S-S are so similar in related compounds being 69. 3 kcal and 70.0 kcal respectively a4, that fission of one without the other would seem unlikely. Fission of the C-N bond also seems very probable on the basis of the bond dissociation energy which is 76 keal/mole (ref. 35). Subsequent reaction of these radicals with the solvent and with each other along with some oxidation could give the observed products. Atom abstraction from the solvent can only be a tentative postulation due to the known stability of the water molecule. However, all products observed can be explained with such a mechanism. Cys-S. radicals from Reaction 5 would give cystine, cysteine or alanine 3-sulphenic acid. The latter unstable acid will either oxidize via alanine 3-sulphinie acid to cysteic acid, or disproportionate, i.e., 2 Cys-SOH-> Cys-SO2H + Cys-SH to give cysteine and alanine 3-sulphinic acid which will oxidize to cysteic acid. Cysteine, cysteic acid and the intermediate alanine 3-sulphinic acid have been identified at both pH values. In alkali, less cysteine is formed than in acid, but this amino acid is known to undergo aerial oxidation in alkaline solution. The Cys. radicals from Reaction 6 would give alanine and serine. The radicals may also rearrange to some extent to give the more stable tertiary free radicals, i.e., NH2-CH-COOH NHi-C-COOH C.,H 2 CH a This radical would give alanine or the following compound NH2-C(CHa)-COOH which / OH would easily decompose to give ammonia and pyruvic acid, i.e., OH O NIt2-C-COOH > NH a + C-COOH J I CH 3 CH a Such a decomposition would account for the presence of some of the ammonia pro- duced and for the presence of pyruvic acid at both pH values; it would also account for larger amount of alanine produced in alkaline solution. The rest of the ammonia appears to be formed by abstraction reactions of the NH 2- radicals, NH2OH has also been found presumably from reaction of the NH~. radicals produced from C-N bond fission. A trace compound identical to the product formed by reacting cysteine with pyruvic acid in the cold is also found at both pH values; slightly more is produced in alkali. This compound is probably the mercaptal CH 3 J Cys-S-C-S-Cys J COOH Biochim. Biophys. At/a, i84 (1969) 345-357
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`THE PHOTOLYSIS OF CYSTINE 355 The Cys-S-S. radicals would form perthiocysteine (Cys-S-SH) and cysteine S-sul- phonic acid (via Cys-S-SOH and Cys-S-SO2H ) by oxidation and abstraction re- actions. Cysteine S-sulphonic acid has been identified in small amounts in acid and alkaline solution. The presence of perthiocysteine has not been established although automatic amino acid analysis of the irradiated solution shows a small peak similar to the one obtained when cystine is treated with Na2S in alkaline solution under N 2 which is a reaction believed to produce perthiocysteine (H. ZAHN, personal communi- cation). Products involving the dimerization and interaction of the initial radicals have been identified and estimated in the irradiated solution. These have formed presum- ably by the appropriate radicals coming together and combining in the solution according to the following reactions. Cys. + Cys-S. -- > Cys-S-Cys Cys-S" + Cys-S-S' -- • > Cys-S-S-S-Cys Cys-S-S" + Cys-S-S" > Cys-S-S-S-S-Cys In alkaline solution these compounds are not detected, probably because the sulphur containing radicals are too hydroxyphilic 36 and will quickly react with the solvent. The absence of a,cd-diaminoadipic acid at both pH values is believed to be due to steric hindrance. Catalin models show that in order to dimerize, two Cys. radicals would have to be correctly aligned. Hence they preferentially react with the solvent or with another radical. An overall reaction scheme is shown in Fig. 9. In acid solution where ammonia is not used in the solvent, some calculation can be made to assess the relative im- portance of the three proposed fissions. Table IV shows the amounts of products formed after a 4-h irradiation in terms of the nitrogen content as a percentages of the total nitrogen available and shows which bonds must be broken to give these h~ Cys • + Cys-S-S * Cys. abstraction Cys-H abstr actioll Cys. ~ Cys-OH Ntt2-CH-COOH ---~NH2~C -COOH CH 2 ~H 3 abstraction [ NH2-~H-COOH eft 3 or NH2-C(OH)-COOH NH 3 + CH3-CO-COOH NH 2. abstraction NH 3 + Nt~2OH colorless deamination products + NH 2 . Cys-SS-eys- Cys-S-S~ abstractior~ Cys-S-SH abstraction Cys-S-S. ~ Cys-S-SOH hv 2 Cys-S' Cys-S" abstraction Cys-SH Cys-S. abstraction Cys!SO H I (o) I (o) Cys -S-SO2H 2Cys-SOH Cys-SO2H (0) (0) Cys-B-SO3H Cys~SO3H- Cys-S-S' + Cys-S' ~ Cys-S-S-S-Cys Cys-S-S" + Cys-S-S'~Cys-S-S-S-S-Cys Cys-S" + Cys' * Cys-S-Cys Fig. 9. The postulated overall reaction scheme for the photolysis of cystine accounting for the proportions of identified products formed. Biochim. Biophys. Acta, 184 (I969) 345-357
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`350 R.S. ASQUITH, L. HIRST TABLE IV CALCULATED RELATIVE IMPORTANCE OF THE BOND BREAKAGE OCCURRING ON EXPOSURE OF CYSTINE Calculation is based on the estimated amounts of decomposition products formed. Product formed I~moles/ml Nitrogen Bond broken Cystine in product as togive product accounted % original N for (%) Cys-SOaH o.658 7.95 Cys-SH 1.31o 15.8o Cys-S-R o.334 4.oo Cys-SO2H Trace Trace Cys-S-R o.334 4.oo Cys-H o.217 2.62 Cys-OH o.268 3.23 CH3-CO-COOH 0.690 8.30 Cys-S-SO3H Trace Trace NH3 I4.O~, NH2OH 14.o Unchanged cystine 0.668 16.2"** S-S 28 C-S 18 C-N 28 * Total NH 3 less that from pyruvic acid formation. ** See RESULTS AND DISCUSSION * ** Residual cystine. compounds. It can be seen that the amount of S-S bond breakage is greater than the amount of C-S bond breakage. Deamination cannot be assessed directly, but it can be assumed that the ammonia produced in excess of that formed during the production of pyruvic acid has been formed from the abstraction reactions of the NH 2. radicals given by C-N fission. The unestimated NH20H also will be formed from reaction of the NH 2. radicals in comparable amounts to ammonia. Using these calculations it seems likely that C-N and S-S bond fission occur to a similar extent. ACKNOWLEDGMENT We thank the Wool Textile Research Council for a grant to one of us (L. H.) and for permission to publish this work. REFERENCES i A. D. McLAREN AND D, SHUGAR, Photochemistry of Proteins and Nucleic Acids, Pergamon Press, (1964). 2 D. F. Louw, L. S. SWART AND P. S. MELLET, S. African J. ,4gri. Sci., 6 (1963) 633. 3 S. RIsI, K, DOSE, T. K. RATHINASAMY AND L. AUGENSTEIN, Photochem. Photobiol., 6 (1967) 423 • 4 J- K. SETLOW, in M. FLORKIN AND E. H. STOTZ, Comprehensive Biochemistry, Vol. 22, Elsevier, Amsterdam, 1967, p. 157. 5 K. DosE, Photochem. Photobiol., 6 (1967) 437- 6 F. G. LENNOX, J. Textile Inst., 51 (196o) T 1193. 7 A. S. INGLIS AND F. G. LENNOX, Textile Res. J., 35 (1965) lO4- 8 A. S. INGLIS, I. H. LEAVER AND F. G. LENNOX, Proc. 3rd Intern. Wool Textile Res, Conf., Paris, r965, Vol. 2, Institut Textile de France, Boulogne, 1965, p. 121. 9 A. S. INGLIS AND F. G. LENNOX, Textile Res. J., 33 (1963) 431. Biochim. Biophys. Acta, 184 (1969) 345-357
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