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`
`JOURNAL OF RADIATION RESEARCH 7-2 47-57 (June 1966)
`
`Radiolysis of Cystine in Aqueous Solution by Gamma Irradiation
`
`ROKUSHIKA, Soji*, GANNO, Shigetake*, SUMIZU, Koichiro*
`
`fli;i
`
`!l!f 11! Ml/:
`and Hiroyuki HAT ANO*
`
`11!l ~ !l!r rJJ fi
`
`(Received, Apr. 16 1966)
`
`ABSTRACT
`
`From several experiments on oxidative radiolysis of cystine in air(cid:173)
`containing aqueous solution a scheme of radiolytic mechanism was
`proposed as follows :
`
`C cystine-S-monosulfone J
`/'
`"\
`H20//
`~H 20 2
`~
`/02H
`cystine ~ cystine hydrogen peroxide -
`cystine
`\"
`monosulfoxide
`
`l
`
`<:"--------:/>--________
`~',.
`--------
`-
`
`✓
`cysteic acid
`/
`✓
`sulfate
`pyruvic_ acid
`{
`ammonia
`Chromatographic behaviors of these intermediate products and the
`radiolytic process were described
`
`-
`
`cysteine
`sulfinic acid
`
`cystine
`disulfoxide
`
`_
`
`{formaldehyde
`carbon dioxide
`
`INTRODUCTION
`
`Several results on radiolysis of amino acids by iomzmg radiation in aqueous
`solution have been reported recently by the authors 1
`)_ From the results a mech(cid:173)
`anism of oxidative radiolysis of amino acid in air-containing solution has been
`proposed 1l. As already well known, sulfhydryl compounds such as cysteine5l,
`* Department of Chemistry, Faculty of Science, Kyoto University, Kyoto, Japan
`
`4
`
`-
`
`47
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`48
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`ROKUSHIKA, GANNO, SUMIZU AND HATANO
`
`•
`
`8
`
`) are more radiosensitive owing to characteristics
`) and glutathione7
`cysteamine6
`of the sulfhydryl group, and sulfur-containing compounds such as cystine9 •10 ), and
`) are also relatively more radiation-labile because of the property of
`methionine11
`). The present paper deals with radiolysis of cystine in air-containing
`the sulfur8
`9
`•
`aqueous solution by relatively small doses of r-irradiation. A mechanism of the
`radiolysis of cystine and its derivatives is discussed.
`
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`
`EXPERIMENT AL
`DL-Cystine used in these experiments was extra pure chromato-
`Materials
`graphically and a product of Tanabe Pharmacological Co., Ltd., Osaka. 2,4-Dinitro(cid:173)
`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
`Ten mM solutions of cystine in 0.1 N sodium hy-
`Irradiation of 6°Co r-rays
`droxide were prepared using redistilled water by an all glass distillator in order to
`avoid some metallo-catalytic decomposition of cystine 12) 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 6°Co Gamma Ray Irradiation Facility13). For irradiation
`of relatively small doses of r-rays, a 100 curie 6°Co Gamma Ray Irradiation Facility
`for Biochemical Research14 ) was used. After the irradiation the tubes were put
`into a dry ice bath for further measurements.
`Amounts of gaseous carbon dioxide were
`Determination of catbon dioxide
`measured manometrically by using a Warburg respiratory manometer.
`Keto acids among carbonyl compounds
`Determination of acidic keto acids
`which were produced in alkaline solutions by the irradiation, were converted to
`2, 4-dinitrophenylhydrazine hydrochloride
`2, 4-dinitrophenylhydrazones by 0.1 N
`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(cid:173)
`metrically at the wave-length 380 mµ in sodium carbonate and bicarbonate solution
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`RADIOL YSIS OF CYSTINE IN SOLUTION BY GAMMA IRRADIATION
`
`49
`
`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 2.0 m
`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 7-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 al15>.
`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(cid:173)
`ification16>: 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(CH2SO3H)COOH, was found to be produced only
`by exposure to larger doses of irradiation. Further progress of the oxidative radio(cid:173)
`lysis made it possible to liberate the sulfuryl compound from the sulfuryl inter(cid:173)
`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
`
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`50
`
`ROKUSHIKA, GANNO, SUMIZU AND HAT ANO
`
`in Fig. 1. On a Dowex-1 column, 0.9 cm
`in dia. and 35 cm in length, retention vol(cid:173)
`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
`Production of
`was presented in Fig. 2.
`pyruvic acid, ammonia and carbon dioxide
`by r-irradiation was also shown in Figs. 2
`and 3.
`Because any other keto acids could not
`be found on paper chromatograms the car(cid:173)
`bonyl product from irradiated cystine was
`found to be only a pyruvic acid of which
`2,4-dinitrophenylhydrazone was separated
`into two spots of cis- and trans-isomers on
`the paper chromatogram.
`
`120
`
`60
`
`90
`
`ml
`Effluent
`Fig. 1 A chromatographic separation of
`cysteine sulfinic acid and cysteic
`acid from the radiolytic products
`of aqueous cystine on the Dowex-1
`column, 0.9 x 35 cm. The 20 mM
`cystine solution were exposed to
`'°Co r rays. The
`2 x 10' R of
`O.Dl N HCI, 0.025 N NaCl buffer
`was used for the elution.
`
`o CySO2H
`• CySO3ll
`x CH3COCOOH
`
`0.5
`
`(l)
`
`·E
`~ 0.4
`v
`c
`~
`E
`~ 0.3
`
`,c 0.2
`c:i ;;;
`
`0.1
`
`104
`
`Dose
`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.
`
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`RADIOLYSIS OF CYSTINE IN SOLUTION BY GAMMA IRRADIATION
`
`51
`
`1.5
`
`.§
`<fl
`>,
`'-J 1.0
`
`~
`
`'F,
`
`~ C
`8.
`~
`E:
`·- 0.5
`"D
`,:;
`~
`
`ONH3
`•CO2
`
`A
`
`B
`
`0
`
`JO'
`
`Dose
`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 aqd 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
`produced from cystine in aqueous
`solution by exposure to 1 x 107 R of 1-
`rays*
`
`I trans form j cis form
`
`Authentic 2,4-DNPH
`pyruvate
`2,4-DNPH of irradiated
`product from cystine
`
`0.70
`
`0.69
`
`0.81
`
`0.83
`
`Rf-values of 2,4-dinitrophenylhy(cid:173)
`drazones of carbonyl compounds pro(cid:173)
`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(cid:173)
`ducts was estimated on a short Am(cid:173)
`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.
`
`* Mean of 5 experiments
`
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`52
`
`ROKUSHIKA, GANNO, SUMIZU AND HAT ANO
`
`Table. 2. G-values of radiolytic products of cystine in aqueous solution
`co,
`CH 3 COCOOH
`
`CySO,H
`
`NH 3
`
`Dose, R
`
`CySO 3H
`
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`
`lxl0'
`5x 106
`2Xl0 6
`lxl0 6
`lxlO'
`
`0.20
`-
`0.46
`-
`1.14
`
`0.62
`-
`2.42
`-
`6.6
`
`1.55
`1.61
`1.47
`1.47
`-
`
`0.15
`0.18
`-
`0.18
`0.11
`
`0.96
`0.75
`-
`0.68
`-
`
`Intermediate products of the radiolysis of cystine in aqueous solution by exposure
`2.
`Acidic and neutral products appeared on long
`to relatively samll doses of r-rays
`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
`
`Effluent
`
`ml
`
`90
`
`120
`
`150
`
`60
`---150cm Column 50'C pH 3.25 0.2 N Na-Citrate------ -
`
`180
`
`210
`
`300
`
`330
`
`360
`
`390
`
`------l-- pH 4.25 0.2 N
`
`Na-Citrate
`
`Effluent
`
`120
`
`150
`
`180
`
`420
`330
`. 90
`ml
`- - - - - - - - - 1 5 0 cm Column 50'C pH 3.25 0.2 N Na-Citrate ~ - - - - - - - - - l - - - - ' - p _H _4_.Z_S_0_.2_A_' _ _
`Na-Citrate
`Fig. 4. Chromatographic separations of radiolytic products of irradiated aqueous cystine
`solutions on the Amberlite CG-120 column 0.9x 150 cm. The cystine solutions were
`analyzed with an automatic amino acid analyzer immediately after exposed to 6°Co
`r-ray for each doses. Upper; 5Xl0 2 roentgens. Bottom; 5Xl05 roentgens. Peak
`this
`in
`is not shown
`E (which will appear where pointed at with an arrow)
`chromatogram.
`
`360
`
`390
`
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`RADIOLYSIS OF CYSTINE IN SOLUTION BY GAMMA IRRADIATION
`
`53
`
`dose Ix 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 mµ was larger than at 570 mµ. 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 I xl04 roentgens and more. Char(cid:173)
`acteristics of the ninhydrin color development of peak A, B, E, F were same as
`that of cystine. The absorbances, E570 mµ, E440 mµ, and E640 mµ were observed to be
`E570 mµ, > E440 mµ > E640 mµ 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
`
`--0- Cystine+peak F product
`-•- Peak B product
`- - - Proposal disappearance of
`cystine
`___ _. Proposal appearance of
`peak F product
`
`100
`
`75
`
`50
`
`25
`
`~ :::
`OJ u
`s..
`~
`.s
`-0 ;;
`~
`
`103
`
`104
`
`106
`
`107R
`
`105
`Dose
`Fig. 5. Formation and degradation of intermediate products in r-irradiated
`cystine solutions by exposure to relatively small doses of r-rays.
`
`102
`
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`54
`
`ROKUSHIKA, GANNO, SUMIZU AND HATANO
`
`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 106 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 2- S- S-CH 2-CH-COOH.
`I
`:1
`I
`NH 2
`0
`NH2
`Chromatographic behaviors of peak B, characteristics of its ninhydrin color de(cid:173)
`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-CH 2-
`I
`NH 2
`S-S-CH2-CH-COOH, which was produced by reaction of hydroperoxyl radical,
`I
`I
`0-0-H NH2
`O 2H, 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-CH 2-S-S-CHi-CH-COOH.
`II
`II
`I
`I
`NH 2
`0 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 O2H radicals.
`R-S-S-R + O2H--R-S-S-R
`I
`0-0-H
`The peroxide is oxidized to cystine monosulfoxide,
`2 R-S-S-R--R-S-S-R+H 2O+1/2 0 2
`II
`0
`
`i
`
`O-O-H
`which is oxidized to disulfoxide.
`R-S-S-R--R-S-S-R+H
`II
`II
`I
`0 0
`0
`Then cystine disulfoxide is decomposed to cystine and cystine sulfinic acid in
`alkaline solution according to Lavine equation17
`3 R-S-S-R+4 NaOH--R-S-S-R+4 R-SO 2H
`II
`II
`0 0
`and cystine sulfinic acid is oxidized to cysteic acid.
`(0)
`R-S02H--R-S0 3H
`As the results on the irradiation of cysteic acid solution, cysteic acid was deaminated
`to give pyruvic acid, carbon dioxide, ammonia and hydrogensulfide.
`
`),
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`RADIOL YSIS OF CYSTINE IN SOLUTION BY GAMMA IRRADIATION
`
`55
`
`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
`
`H 2N-C-COOH+0 2H
`I
`-
`CH 2S03H
`HN =C-COOH +OH+ H 20 2
`I
`CH2S03H
`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
`though acetaldehyde has not yet
`and carbon dioxide CH3COCOOR-+CH3CRO+C02
`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.
`0
`0-0-R
`S--S
`II
`I
`I
`S -S
`S -S
`H 2C
`I
`I
`I
`I
`I
`CH 2
`R 2C
`CR2
`R 2C
`H 2NCR HCNR 2
`I
`I
`I
`I
`I
`I
`H2NCR RCNR 2
`R2NCR RCNR 2
`COOR
`ROOC
`I
`I
`I
`I
`COOR
`HOOC
`COOR
`ROOC
`
`CR 2
`
`!
`
`cystine
`
`(F) cystine
`monosulfoxide
`
`NH3+S~
`+
`CR3COCOOH
`i
`CO2
`
`pyruvate
`
`-
`
`S-03H
`I
`CH2
`I
`HCNH2
`I
`COOH
`
`--
`
`--
`
`cysteic
`acid
`
`cysteine
`sulfinic acid
`
`(A) cystine
`disulfide
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`56
`
`ROKUSHIKA, GANNO, SUMIZU AND HAT ANO
`
`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, Band E were disappeared and only peak F was found to appear dur(cid:173)
`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 reaction18
`) were proposed.
`
`-0 - CyS+peak F product
`- • - Peak E product
`-x- CySOi H -'-CySOJ H
`
`100
`
`~
`·- 50
`
`2
`
`•; ..,
`
`10 days
`
`Time after irradiation
`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.
`
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`RADIOLYSIS OF CYSTINE IN SOLUTION BY GAMMA IRRADIATION
`
`57
`
`or
`
`R-S-S-R +1/2 H2
`I
`0 2H
`R-S-S-R+H20
`I
`02
`
`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.
`
`I . On radiolysic
`
`REFERENCES
`( 1) Hatano, H. (1961) Oxidative radiolysis of amino acids, peptides and proteins in aq(cid:173)
`ueous solutions by gamma irradiation. Bull. Inst. Chem. Res., Kyoto Univ., 39: 120.
`( 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.
`f. Rad. Res.,
`1: 28.
`( 3) Hatano, H., Ganno, S. and A. Ohara (1963) Radiation sensitivity of amino acids in
`solution and protein to gamma rays. Bull. Inst. Chem. Res., Kyoto Univ., 41: 61.
`( 4) Hatano, H. and S. Rokushika (1963) Oxidative deamination of several amino acids in
`aqueous solution by gamma irradiation. ibid., 41: 76.
`( 5 ) Hatano, H. (1962) Studies of radiolysis of amino acids and proteins.
`oxidation of sulfhydryl groups. J. Rad. Res., 1: 23.
`( 6) Shapiro, B. and L. Eldjarn (1955) The effects of ionizing radiation on aqueous solutions
`of cysteamine and cystamine. Rad. Res., 3: 255.
`( 7) Littman, F. E., Carr, E. M. and A. P. Brady (1957) The action of atomic hydrogen
`on aqueous solutions. I. Effect on silver, cysteine and glutathione solutions.
`ibid., 7: 107.
`( 8) Kinsey, V. E. (1935) The effect of X-rays on glutathione.
`f. Biol. Chem., 110: 551.
`( 9) Markakis, P. and A. L. Tappe! (1960) Products of r-irradiation of cysteine and cystine.
`f. Am. Chem. Soc., 82: 1613.
`(10) Grant, D. W., Mason, S. N. and M.A. Link (1961) Products of the r-radiolysis of
`aqueous cystine solutions. Nature, 192: 352.
`(11) Shimazu, F., Kumta, U.S. and A. L. Tappe! (1964) Radiation damage to methionine
`and its derivatives. Rad. Res., 22: 276.
`(12) DE Marco, C., Colletta, M. and D. Cavallini (1963) Cystine cleavage in alkaline medium.
`Arch. Biochem. Biophys., 100: 51.
`(13) Okamoto, S., Nakayama, Y. and K. Takahashi (1959) The two kilocurie cobalt-60
`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(cid:173)
`matography of amino acids Anal. Chem., 30: 1190.
`(16) Rokushika, S., Ohara. A., Anma T., Sumizu, K. and H. Hatano to be published.
`(17) Toennies, G., and T. F. Lavine (1936) The oxidation of cystine in non-aqueous media.
`V. Isolation of a disulfide of l-cystine. J. Biol. Chem., 113: 571.
`(18) Shapiro, B. and L. Eldjarn (1955) The mechanism for the degradation of cystamine
`by ionizing radiation. Rad. Res., 3: 393.
`
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