Radiolysis of Cystine in Aqueous Solution by Gamma Irradiation * ROKUSHIKA Soji *GANNO Shigetake *SUMIZU Koichiro

Radiolysis of Cystine in Aqueous Solution by Gamma Irradiation

ROKUSHIKA Soji *GANNO Shigetake *SUMIZU Koichiro*

*六鹿宗治 *雁野重威 *隅水〓一郎

iroyuki HATANO *H

波多野* 博行

(Received, Apr. 16 1966)

ABSTRACT

From several experiments on oxidative radiolysis of cystine in air containing aqueous solution a scheme of radiolytic mechanism was proposed as follows :

Chromatographic behaviors of these intermediate products and the radiolytic process were described

INTRODUCTION

Several results on radiolysis of amino acids by ionizing radiation in aqueous solution have been reported recently by the authors' -4>. From the results a mech anism of oxidative radiolysis of amino acid in air-containing solution has been proposed'). As already well known, sulfhydryl compounds such as cysteine5), *Department of Chemistry , Faculty of Science, Kyoto University, Kyoto, Japan cysteamine6) and glutathione7'8) are more radiosensitive owing to characteristics of the sulfhydryl group, and sulfur-containing compounds such as cystine9'10), and methionine11> are also relatively more radiation-labile because of the property of the sulfur8'9). The present paper deals with radiolysis of cystine in air-containing aqueous solution by relatively small doses of r-irradiation. A mechanism of the radiolysis of cystine and its derivatives is discussed.

EXPERIMENTAL Materials DL-Cystine used in these experiments was extra pure chromato graphically and a product of Tanabe Pharmacological Co., Ltd., Osaka. 2,4-Dinitro phenylhydrazine, methylcellosolve, citric acid, sodium hydroxide and hydrochloric acid were obtained from Nakarai Chemicals Co., Ltd., Kyoto. Water used in these experiments was prepared through a ion-exchange resin column and distilled twice by an all glass distillaton apparatus in order to avoid catalytic decomposition of cystine by a small amount of heavy metals in water. For all glass tubes, ampoules, and vessels arrangements were also made in order to avoid the decomposition by impurity of heavy metals.

METHODS Irradiation of 60Co r-rays Ten mM solutions of cystine in 0.1 N sodium hy droxide were prepared using redistilled water by an all glass distillator in order to avoid some metallo-catalytic decomposition of cystine12) and put into tubes (12 mm. in dia. and 100 mm. in length). The tubes were sealed and irradiated at room temperature by exposure to r-irradiation with a dose rate of 1.2 x 105 roentgens per hour in a Two Kilo-curie 60Co Gamma Ray Irradiation Facility13). For irradiation of relatively small doses of r-rays, a 100 curie 60Co Gamma Ray Irradiation Facility for Biochemical Research 14) was used. After the irradiation the tubes were put into a dry ice bath for further measurements. Determination of carbon dioxide Amounts of gaseous carbon dioxide were measured manometrically by using a Warburg respiratory manometer. Determination of acidic keto acids Keto acids among carbonyl compounds which were produced in alkaline solutions by the irradiation, were converted to 2, 4-dinitrophenylhydrazones by 0.1 N 2, 4-dinitrophenylhydrazine hydrochloride reagent. The hydrazone mixtures were extracted with ethyl acetate and reextracted with 0.1 N sodium carbonate and sodium bicarbonate solution. The alkaline solutions thus obtained were made acidic by addition of hydrochloric acid, and extracted again with ethyl acetate. Qualitative analyses of the hydrazones of keto acids were carried out with a paper chromatography using a developing solvent of ethanol: n-butanol : 0.1 N sodium bicarbonate (10: 10: 3 v/v) and No. 50 Toyo filter papers. Quantitative determination of the keto acid hydrazones was performed spectrophoto metrically at the wave-length 380 mµ in sodium carbonate and bicarbonate solution by using Hitachi EPU-2 spectrophotometer. Determination of volatile organic acids and aldehydes A gaschromatograph, Yanagimoto Model GCF-100, was used for measurements of volatile radiolytic products from the cystine solutions. For the chromatographic estimation, a 2.Om column in which C-22 coated with polyethyleneglycol 1500 was filled, was operated at 130°C with a carrier nitrogen gas at a running rate of 30 ml per min. The results were recorded automatically. Analysis of ninhydrin positive products An automatic amino acid analyzer, Hitachi Model KLA-2, was used for determination of ninhydrin positive compounds produced from cystine in aqueous solution by the irradiation. For chromatographic separation of cysteic acid and cysteine sulfinic acid, a Dowex-1 x 8, 200 to 400 mesh, column (0.9 cm in dia. and 35 cm in length) was operated at 50°C and an eluting rate of 30 ml per hour with an eluting buffer of 0.01 N hydrochloric acid and 0.025 N sodium chloride. Acidic and neutral compounds were separated on an Amberlite CG 120 x 8,325 to 400 mesh column, (0.9 cm in dia. and 150 cm in length) by running 0.2 N citrate buffers, pH 3.28 and 4.25, at a rate of 30 ml per hour. Ammonia and basic compounds were analyzed by the procedure as follows: after r-irradiation of the sealed test tubes containing the amino acid materials, 1 N hydrochloric acid was added air-tightly into the test tubes by a syringe through a rubber stopper until pH 1 to 2. The acidic solution containing the radiation products was analyzed chromatographically using an Amberlite CG 120, 400 mesh, column (0.9 cm in dia. and 15 cm in length) and a 0.35 N citrate buffer, pH 5.28, under the conditions described by Spackman et ally Analysis of amines Amines in the radiation products were analyzed by a new method of procedure for analysis of basic amino acids with the following mod ification") : three kinds of buffers, 0.35 N citrate, pH 5.28, 0.025 M borate, pH 8.02, and 0.20 M salycilate pH 11.08, were employed successively for the amine analysis at 50°C and a running rate of 30 ml per hour. Specimens of cysteine sulfinic acid and cystine sulfoxide were prepared by oxidation of cystine with perbenzoic acid.

RESULTS AND DISCUSSION 1. Radiolytic oxidation of cystine to cysteine sulfinic acid and to cysteic acid. Cys tine was decomposed oxidatively in the air-containing solutions irradiated with relatively large doses from 104 to 107 roentgens. Among the radiation products after exposure to several kilo roentgens, cysteine sulfinic acid, H2NCH (CH2SO2H) COOH, appeared, and cysteic acid, H2NCH(CHZSO3H)COOH, was found to be produced only by exposure to larger doses of irradiation. Further progress of the oxidative radio lysis made it possible to liberate the sulfuryl compound from the sulfuryl inter mediates producing carbonyl compounds such as pyruvic acid and end products, carbon dioxide and ammonia. The amounts of these radiation products were increased linearly with increasing radiation doses. A chromatogrphic separation of cysteine sulfinic acid and cysteic acid was shown in Fig. 1. On a Dowex-1 column, 0.9 cm in dia. and 35 cm in length, retention vol umes were found to be 60 ml for cysteine sulfinic acid and 100 ml for cysteic acid as shown in Fig. 1. The formation of the amino sulfuric compounds with the increase of r-ray doses was presented in Fig. 2. Production of pyruvic acid, ammonia and carbon dioxide by r-irradiation was also shown in Figs. 2 and 3. Fig. 1 A chromatographic separation of cysteine sulfinic acid and cysteic Because any other keto acids could not acid from the radiolytic products be found on paper chromatograms the car of aqueous cystine on the Dowex-1 column, 0.9 x 35 cm. The 20 mM bonyl product from irradiated cystine was cystine solution were exposed to found to be only a pyruvic acid of which 2 x 106 R of 60Co r rays. The 2,4-dinitrophenylhydrazone was separated 0.01 N HCI, 0.025 N NaC1 buffer was used for the elution. into two spots of cis and trans-isomers on the paper chromatogram.

Fig. 2. Formation of cysteine sulfinic acid, cysteic acid and pyruvic acid. Solution of cystine, 10 mM was irradiated in 1.2 x 105 R/h dose rate at room temperature. Before analysis of the irradiation products, except pyruvic acid, this solution was adjusted to pH 2.2. Amount of pyruvic acid was measured by the Katsuki's method as described in text. A curve represents yields of cysteine sulfinic acid. B curve ; cysteic acid and C curve ; pyruvic acid. Fig. 3. Formation of ammonia and carbon dioxide. The solution of cystine , 10 mM, was irradiated in 1.2 x 105 R/h dose rate at room temperature. Ammonia was determined chromatographically using an automatic amino acid analyzer . Carbon dioxide was measured by the manometric method using Warburg's apparatus. A curve represents yield of ammonia. B curve : carbon dioxide.

Table 1. Rf-values of carbonyl compounds Rf-values of 2,4-dinitrophenylhy produced from cystine in aqueous drazones of carbonyl compounds pro solution by exposure to 1 x 10' R of r rays* duced in r-irradiated cystine solutions on the paper chromatograms were presented in Table 1. Carbon dioxide decarboxylated from pyruvic acid was estimated and presented in Fig. 3. Ammonia among irradiation pro

* Mean of 5 experiments ducts was estimated on a short Am berlite CG-120, 400 mesh, column being eluted at the retention volume of 80 ml, when no basic products such as basic amino acids and amines were found on the column. The amounts of ammonia determined at various doses of r-irradiation were given in Fig. 3. G-values of these irradiation products from cystine solutions were presented in Table 2. Table. 2. G-values of radiolytic products of cystine in aqueous solution

2. Intermediate products of the radiolysis of cystine in aqueous solution by exposure to relatively smmll doses of r-rays Acidic and neutral products appeared on long Amberlite CG-120 columns were shown in Fig. 4. Five unknown products on the chromatograms were found to be varied by irradiation doses, named as peak A, B, D, E and F. Peak B appeared by relatively small doses of r-rays showed a maximum yield at the dose of 1 x 104 roentgens, and decreased to disappear by larger doses at the

Fig. 4. Chromatographic separations of radiolytic products of irradiated aqueous cystine solutions on the Amberlite CG-120 column 0.9 x 150 cm. The cystine solutions were analyzed with an automatic amino acid analyzer immediately after exposed to 60Co r-ray for each doses. Upper ; 5 x 102 roentgens. Bottom ; 5 X 105 roentgens. Peak E (which will appear where pointed at with an arrow) is not shown in this chromatogram. dose 1 x 106 roentgens. In replace of the disappearance of the peak B from the chromatogram, peak A was observed to appear and to increase by larger doses of 7-rays. Cystine sulfoxides (monosulfoxide and disulfoxide) were eluted at the same as peak A and cystine of the long Amberlite CG-120 column. Peak D was found to be produced in the solutions, and to have abnormal ninhydrin colour of which absorbance at 440 m,-z was larger than at 570 m,a. Peak C seemed to be due to an impurity of cystine because it appeared on the chromatogram of cystine solution before irradiation and the production did not depend upon the doses of irradiation. Peak E appeared on the chromatogram between peak A and cystine, will be described below. Peak F appeared on the chromatogram at the same position as that of cystine, and was produced by the dose 'of 1x104 roentgens and more. Char acteristics of the ninhydrin color development of peak A, B, E, F were same as that of cystine. The absorbances, E570m,, E440mu, and E640mp were observed to be E570mu, > E440mp > E640mu for these peaks at the three wave lengths contrary to other amino acids. A relation of the appearance and the disappearance of cystine, peak B and F, was shown in Fig. 5. The amount of peak B increased between a region

Fig. 5. Formation and degradation of intermediate products in r-irradiated cystine solutions by exposure to relatively small doses of r-rays.

O1 11 -O-H O which is oxidized to disulfoxide. R-S-S-R-->R-S-S-R + H

I li II 1 NH2 O 0 NH2 A radiolytic mechanism of cystine in air-containing aqueous solution is proposed from the results mentioned above as follows : cystine, R-S-S-R, is oxidized to cystine hydroperoxide by 02H radicals. R-S-S-R + 02H-- R-S-S-R OI -O-H The peroxide is oxidized to cystine monosulfoxide, 2 R-S-S-R---•R-S-S-R + H20+1/2 02

I II I NH2 O NH2 Chromatographic behaviors of peak B, characteristics of its ninhydrin color de velopment as like as cystine, and its high radiosensitivity and unstability showed that th peak B product was to be cystine S-hydrogen peroxide, HOOC-CH-CHZ NHI 2 S-S-CHZ CH-COOH, which was produced by reaction of hydroperoxyl radical, 0-O-H NH2 02H, with cystine in oxygen-containing solution irradiated by relatively small doses. The cysteine-S-hydrogen peroxide may be oxidized to cystine monosulfoxide because it is very unstable in the aqueous solution. Peak A appeared to be cystine disulfoxide HOOC-CH-CH2-S-S-CH2-CH-COOH.

from 102 to 104 R doses from which decreased to 106 R dose. From 104 to 105 R doses peak F was increased and from 105 to 10I R decreased. From these results it was presumed that at the 104 R dose the 50% cystine converted to the peak B product and the disappearance of cystine and the peak B product was coincident. From these result and comparison with a synthetic specimen of cystine sulfoxide which was eluted at the same on chromatogram, the peak F compound was proposed to be identical with cystine monosulfoxide HOOC-CH-CH, S S -CHZCH-COOH.

O O O Then cystine disulfoxide is decomposed to cystine and cystine sulfinic acid in alkaline solution according to Lavine equations'), 3 R-S-S-R + 4 NaOH-- R-S-S-R + 4 R-SO2H OII OII and cystine sulfinic acid is oxidized to cysteic acid. (O) R-S02H--R-SO3H As the results on the irradiation of cysteic acid solution, cysteic acid was deaminated to give pyruvic acid, carbon dioxide, ammonia and hydrogensulfide. In the course of pyruvate formation no production of alanine and glycine was found resulting pyruvate derived directly from cysteic acid. Because no other keto acids occurred in the course of the decomposition, decarboxylation, deamination of cysteic acid seemed to occur simultaneously. and desulfurylation

Occurrence of free sulfur was proved at a large dose irradiation of 107 R when irradiated mixture is containing white precipitate. It is confirmed from the results that when a lead acetate or sulfate reagent was added to the radiation mixture, blackish brown precipitate appeared in the solution in which hydrogen sulfide is also found to exist. Pyruvate may further decarboxylated to produce acetaldehyde and carbon dioxide CH30OCOOH--CH3CHO+CO2 though acetaldehyde has not yet been observed on the gas chromatogram. Because no occurrence of cystamine and cysteamine was observed, cystine might not be decarboxylated directly to produce carbon dioxide. From the results mentioned above, a radiolytic mechanism of cystine in air-containing aqueous solution was proposed as shown below. 3. After effects in an alkaline cystine solution When an alkaline soltuion of cystine was irradiated by r-rays and allowed to stand at 5°C or room temperature for several days, it was observed an after effect which depended on irradiation conditions and standing periods. After exposuring to a 5 x 105 R dose and standing for 1 hour at room temperature no effect was observed but in the r-irradiated cystine solution almost all peak F product disappeared and a new peak E appeared between peak A and cystine peak as shown in Fig. 6. An amount of the peak E product was almost as same as that of the peak F product. After standing for 3 days similar results were observed except to that a small amount of cystine seemed to change to cystine sulfoxide according to the Lavine's reaction. After allowing to stand for 10 days at 5°C, peaks A, B and E were disappeared and only peak F was found to appear dur ing which cysteine sulfinic acid and cysteic acid was increased gradually. It was presumed that during allowing to stand for 3 days the peak F product may be recovered back to cystine by the after effect. The peak E product may be derived from both cystine and cystine monosulfoxide not immediately, but after standing for 1 day. From these observations contributions of both relatively stable entities such as hydrogen peroxide and unstable radicals to formation of the peak E product by Shapiro's reaction 18) were proposed.

Fig. 6. After effect of irradiated aqueous cystine solutions. After exposed for 5 x 105 R dose and allowed to stand for 1, 3 and 10 days at 5°C the solutions were analyzed with an amino acid analyzer. Quantitative estimation of peak E and peak F products were done by assuming that colour yields of those products with ninhydrin reagents are equivalent to that of cystine. The product may be cystine-S-monosulfone.

From the fact that the recovery of cystine from cystinemonosulfoxide formed by irradiation was observed when irradiated solution was allowed to stand for one day after the lower dose of irradiation, such as 5 x 102 R, it is seemed possible that radiolysis process may stop at the step in which peroxide is formed during the irradiation, and that the process between peroxide and cystine is reversible.

REFERENCES

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(2) Hatano, H. (1962) Studies on radiolysis of amino acids and proteins. II. On radiolytic deamination of amino acids in aqueous solutions by gamma irradiation. J. Rad. Res., 1: 28.

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gamm-ray irradiation faculty. Bull. Inst. Chem. Res., Kyoto Univ., 37: 299. (14) Hatano, H., Ganno, S., Sumizu, K., Kimura, N., Ohnishi, S., Imai, T. and T. Tsuzuki

(1965) ibid, 43: in press. (15) Stein, W. H. and S. Moore (1958) Automatic recording apparatus for use in the chro matography of amino acids Anal. Chem., 30: 1190.

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