Hatano, Hiroyuki

Rokushika, Souji

Kyoto University Research Information Repository

TitleOxidative Deamination of Several Amino Acids in AqueousSolution by Gamma Irradiation (Special Issue on Physical,Chemical and Biological Effects of Gamma Radiation, IV)Author(s)Hatano, Hiroyuki; Rokushika, SoujiCitationBulletin of the Institute for Chemical Research, KyotoUniversity (1963), 41(1): 76-82Issue Date1963-03-30URL http://hdl.handle.net/2433/75941RightType Departmental Bulletin PaperTextversionpublisherKyoto University     Oxidative Deamination of Several Amino Acids in         Aqueous Solution by Gamma Irradiation*                Hiroyuki HATANO and Souji  ROKUSHIKA**            (Department of Chemistry, Faculty of Science, Kyoto University)                           Received December 25, 1962    A general overall reaction of oxidative deamination of amino acid in aqueous solu- tion, H2NRCHCOOH---> NH3+RCOCOOH, was characterized on leucine, a-aminobutyric  acid and oxaloacetic acid after exposure to -irradiation. Keto acids corresponding to  the parent amino acids were produced by Y-irradiation and determined quantitatively by  chromatography and spectroscopic measurements after being derived to 2, 4-dinitrophenyl- hydrazones.     Some spectroscopic and chromatographic properties of the keto acid hydrazones were  reported here continueing to the previous reports.                           INTRODUCTION    Radiolytic reactions of amino acids in solution were studied by many in-vestigators1-G' and, recently, oxidative radiolysis of amino acid, peptides and proteins was studied by Hatano7 '0' and Garrison et al.11'. One of the major reactions induced in amino acids in solution by radiation is oxidative deamina-tion to arise ammonia and keto acid corresponding to the parent amino acid under an aerobic condition in terms of a general overall reaction : H2NRCHCO0H+1/2 03 —> NHa+RCOCOOH    In the previous papers) the author described that when the each aqueous solution of alanine, glutamic acid and valine was irradiated with 1--rays the corresponding keto acid, pyruvic acid, a-ketoglutaric acid and a-ketoisovaleric acid respectively, were produced from the parent amino acid accompanying by liberation of ammonia as follows : H2NCH000H+1/2 02 —> NH3 + CH3000OOH CH3 H2NCH000H+1/2 Oz -> NH3 + HOOCCH2CH2COCOOH                   CH2 CH2OOOH H2NCH000H+1/2 02 —> NH3 + (CH3)2CH000OOH CH(CH3)z    The further study10' showed the several ultraviolet spectra of 2, 4-dinitro- * The material in this paper was presented at the 4th Symposium on Isotope in Japan,    May 1962, The Proceedings, R-35, p. 401. **rY~'tdf17, 7"l-  ( 76 )                    Oxidative Deamination of Several Amino Acids  phenylhydrazones of many keto acids proposed to be produced from irradiated amino acids in solution such as glyoxalic acid produced from glycine, oxaloace-tic acid from aspartic acid, isopropylpyruvic acid from leucine, phenylpyruvic acid from phenylalanine, (3-hydroxyphenylpyruvic acid from tyrosine, (3-hydro-xypyruvic acid from serine, a-keto-/3-hydroxybutyric acid from threonine, /3-mercartopyruvic acid from cysteine, a-keto-r-inethiolbutyric acid from methionine, a-keto-8-guanidinovaleric acid from arginine, a-keto-e-aminocaproic acid from lysine, /3-imidazolylpyruvic acid from histidine, Q-indolpyruvic acid from trypto-phan and a-keto-8-aminovaleric acid from proline.    In this paper more further experiments were presented on separation and characterization of oxaloacetic acid, isopropylpyruvic acid, and a-ketobutyric acid, which were produced from r-irradiated aspartic acid, leucine, and a-amino-butyric acid respectively, in aqueous solution under the aerobic condition.                           EXPERIMENTAL    Materials. Leucine and aspartic acid used in this experiments were a pro-duct of Ajinomoto Co. Ltd., Tokyo, a-aminobutyric acid was that of Tokyo Kasei Co. Ltd., Tokyo. All were chromatographic pure grade. Authentic specimen of isopropylpyruvic acid, methylethylpyruvic acid and oxaloacetic acid was pre-pareded and kindly supplied by Dr. H. Katsuki of Kyoto University, Kyotole'. Reagent 2, 4-dinitrophenylhydrazine was at a concentration of 1 mg per ml in 2 N hydrochloric acid.    Methods. Irradiation of r-rays : A two-kilocurie cobalt-60 r-ray irradiation facility13' was used for r-irradiation. The dose rate was approximately 1.4 X 105r. per hour.    Spectrophotometric measurements of keto acid 2, 4-dinitrophenylhydrazones : The keto acid 2, 4-dinitrophenylhydrazones was prepared by the method described in the previous papers3"11'. Spectrophotometric measurements were carried out on the preparation of the keto acid derivatives using a Beckman DU spectro-photometer and a Hitachi EP3-2 selfrecording spectrophotometer according to the procedure of the previous papers8,11' .    Chromatography of the keto acid 2, 4-dinitrophenylhydrazones : Paperchro-matograpy of the keto acid 2, 4-dinitrophenylhydrazones were performed accord-ing to the method of Katsuki12) as follows : Toyo filter paper No. 51 was immersed into a mixture of ether and diethylphthalate (80 : 20 v/v) and dried in air as soon as possible. The treated filter paper was used for reverse phase chroma-tography on which a 0.1 N sodium bicarbonate solution was used as a developing solvent. For ordinary paperchroinatography a usual method described in the pre-vious report"' was used. An untreated Toyo filter paper No. 51 and a developing solvent of n-butanol, ethanol and 0.1 N sodium carbonate (4 : 1 : 2, v/v) or filter paper treated with a 0.1 N Na2CO3 solution and a developing solvent of n-butanol, ethanol, 0.1 N Na2CO3 (10 : 3 : 10, v/v) were used in these experiments.    Quantitative determination of the a-keto acids was carried out successfully using Hyflo-Super-Cel column chromatography and spectrophotometric measure-                             (77 )                     Hiroyuki HATANO and Souji  ROKUSHIKA ments by the method described in the previous papers8' .                            RESULTS    (1) Ultraviolet and visible absorption spectra of 2, 4-dinitrophenylhydrazones of the a-keto acids. After exposure to r-irradiation of a 320 kiloroentgen dose the aqueous solutions of a-aminobutyric acid, isoleucine, leucine and aspartic acid were reacted with the 2, 4-dinitrophenylhydrazine reagent to produce the hydrazones of carbonyl compounds. In the resulted mixture it was found that produced a-ketobutyric acid, isopropylpyruvic acid, and oxaloacetic acid which was followed by a secondary product, pyruvic acid, were derived respectively to the hydrazones.    Fig. 1 showed the ultraviolet and visible absorption spectra of 2, 4-dinitro-phenylhydrazones of a-ketobutyric acid and isopropylpyruvic acid, which were .6-------------------------------------------------------------------------------    AB          a •a .4-/~'--  ci?Z Qs)s^6a6/   IU O)//^^^~`/a^.``,.       400`500 600 700 400 500 600 700 Wavelength in m    Fig. 1. Absorption spectra of 2, 4-dinitrophenylhydrazones of the a-keto acids produced     from the 1-irradiated solutions of the parent amino acids, measured in alkaline solu-     tions. A: The hydrazone of a-ketobutyric acid produced from 1-rayed a-aminobuyric acid.     B : That of isopropylpyruvic acid produced from 1-rayed leucine, a : cis-isomer in     0.5 N Na2CO3; a' : trans-isomer in 1 N Na2CO3 ; b : cis-isomer in 0.5 N Na2CO3 and 0.5 N NaOH ; b' : trans-isomer in 0.5 N Na2CO3 and 0.5 N NaOH.     The 10 mM solutions of the amino acid were irradiated with 1-rays of 320 kr. dose.                                                                         L.34, O,1 ON 35 1th P..- q:h i mu    Fig. 2. Absorption spectra of 2, 4-dinitrophenylhydrazones of 1-irradiated isoleucine     and of an authentic specimen (A) of methylethylpylpyruvic acid (B) in an alkaline      solution. ( 78 )                   Oxidative Deamination of Several Amino Acids produced from the parent amino acids respectively. Methylethylpyruvic acid, however, failed to be showed in the irradiated isoleucine solution as shown as Fig. 2.    (2) Paperchromatography of  2,4-dinitrophenylhydrazones of the keto acids. Good separation and identification of cis- and trans- isomers of isopropylpyruvic acid and a-ketobutyric acid 2, 4-dinitrophenylhydrazones were carried out using the ordinary and the reverse phase chromatographies. Oxaloacetic acid deriva-tive arise no isomers of the derivatives. Rf values of the paperchromatography were tabulated in Table 1.    Table 1. Paperchromatographic characterization of keto acid 2, 4-dinitrophenylhydra-    zones which were derived from Y-irradiation products of amino acids in aqueous solu-      tions. 2, 4-dinitrophenylhydrazoneRf Ordinary method Reverse phase method   Pyruvic acid authentic trans-isomer.45.56  Pyruvic acid authentic cis-isomer.53.35  Oxaloacetic acid authentic.37.71   Irradiated aspartic acid.38, .44, .52 .75, .55 Isopropylpyruvic acid authentic trans-isomer .60—   Isopropylpyruvic acid authentic cis-isomer .70— Irradiated leucine.57, .74** —   a-Ketobutyric acid authentic trans-isomer* .48.64 a-Ketobutyric acid authentic cis-isomer*.56.33 Propion aldehyde authentic.28—   Irradiated a-aminobutyric acid.55, .44, .29 .62, .37       All Rf values were mean obtained from 15-20 experiments.    * A solvent, n-butanol : ethano1=10 : 3: 10 and filter papers treated with a 0.01/V       NaHCO3 solution were used. Spot of propion aldehyde derivative coloured brown,       different from other ordinary colour of yellow.    ** More two spot appeared at Rf .33 and .22 which were unknown.    *** More one spot was found at Rf .52, which was also uncharacterized.    (3) Column chromatography of 9, 4-dinitrophenylhydrazones of the keto acid. Quantitative separation and isolation of cis- and trans-isomers of the keto acid derivatives were performed by the column chromatography using a Hyflo-Super-Cel column. Fig. 3 shows the chromatograms of the keto acid derivatives. A secondary product from oxaloacetic acid was identified with pyruvic acid of which 2, 4-dinitrophenylhydrazone have the cis- and trans- and trans-isomers.                           DISCUSSION    In the previous paper it was proposed that the radiolytic deamination of amino acid to produce the corresponding keto acid should be proceed through a radical mechanism of indirect action of radiation as follows : H2NCHCOOH+OH —p H2NCCOOH RR ( 79 )                     Hiroyuki HATANO and  Souji ROKUSHIKA 6o  AL/ Ehent/2 pvrwic aei.i oerivatives  ttunsmer v ,laxer,,-ac:ddermmome     iI                 c]2                                 .4      .2'All  Ol7 6 Ay/hectic isopeopyruvic mcia derivatives               F B           q, ~~il'- na,               0 0 2-ffIQ flfuluenl in ml 20 90 60 80 /00 /20 /90 /60 /80 200 (--'Ethyl acetate (mlfured.vith oil VA CC3and -----n-fiutano6-E1hyl acetate ( both buffered vvitir--1 80/N Adt1CO3011 Na,CO, omel 00/N Na1-1COd)-1:1    Fig. 3. Column chromatographic separation of keto acid 2, 4-dinitrophenylhydrazones     which were derived from I-irradiation products of amino acids in aqueous solutions. A : A chromatogram of 2, 4-dinitrophenylhydrazones of radiation products produced     from an irradiated aspartic acid solution.     Peak I : cis-isomer of pyruvic acid 2, 4-dinitrophenylhydrazone     Peak II: uncharacterized     Peak III : trans-isomer of pyruvic acid 2, 4-dinitrophenylhydrazone     Peak IV : oxaloacetic acid 2, 4-dinitrophenylhydrazone B: That of an irradiated leucine solution.     Peak I : cis-isomer of isopropylpyruvic acid 2, 4-dinitrophenylhydrazone     Peak II : trans-isomer of isopropylpyruvic acid 2, 4-dinitrophenylhydrazone     Peak III: uncharacterized      Hyflo-Super-Cel column, 9 mm X 210 mm was used. H2NCCOOH+O2H —> HN=CCOOH+H2O2 RR HN=CCOOH+H2O —> NH3 + RCOCOOH R Three keto acids had been characterized and results obtained in this experi-ment added further four examples to this radiolytic oxidation of amino acid.    Each amino acid would be perhaps deaminated by the indirect action of r-radiation through the mechanism over all as follows : H2NCHOOOH                 CH2 —* NI-13+CH3CH2OOCOOH                   CH3 H2NCHOOOH                                        CH3                CH2---->NH3 +>CHCH2000QOH CH3/                 CH /                 CH3 CH3 H2NCHOOOH                                           CH3            CH>CH3 +>CHCOCOOH        / \CH3CH2/                 CH3 CH2                     CH3 ( 80 )                    Oxidative  Deamination of Several Amino Acids      Oxaloacetic acid, however, was further rendered to decomposition owing to  its expraordinary instability arising pyruvic acid and carbon dioxide spontaneously  as follows : H2NCH000HCOCOOH CH2 NH3 + CH2         COOHCOOH                COCOOHCH2—> CH3COCOOH + CO2               COOH  The pyruvic acid was confirmed by the chromatographic evidences. Methylethyl- pyruvic acid proposed above to be produced from irradiated isoleucine could not  detected spectroscopically and chromatographically in this experiment. Though  the identification of many other keto acids was not always be successful easily  owing their instability and radiosensitivity, it appears to be certainly in general  that amino acid may be deaminated oxidatively by the radiation products of  water in y-irradiated aqueous solutions to produce the corresponding keto acid  and ammonia according to the following over all reaction : H3NRCHCOOH —+ NH3 + RCOCOOH      Furthermore, these keto acids may be changed to the corresponding aldehydes  by spontaneous decarboxylation or a radiation-induced reaction, as follows :                 RCOCOOH —* RCHO + CO2  Propion aldehyde produced from a-ketobutyric acid was detected on the paper- chromatogram of irradiation products of a-aminobutyric acid, as presented in  Table 1, according to the following reaction : H2NCHCOOH CH2 --> NH3 + CH3CH2COCOOH                     CH3 CH3CH2COCOOH --* CH3CH2CHO      In the peptides and proteins described previously, resulted mixtures of these  keto acids and aldehydes have been investigated using the methods of spectro-  scopic measurements and chromatographic separation of 2, 4-dinitrophenylhydra-  zones of these carbonyl compounds7"10. Volatile aldehydes and other fragmental  compounds may be detected by a gaschromatographic procedure.                          ACKNOWLEDGMENTS      The authors wish to express their thanks to Prof. S. Shimizu, of Kyoto  University for his kind arrangement of gamma irradiation. The authors also  thank Prof. S. Tanaka and Mr. S. Ganno for their useful discussion.                             REFERENCES    (1) W. H. Dale, J. V. Davies and C. W. Gilbert, Biochem. J. 45, 93 (1949). ( 81 )                     Hiroyuki HATANO and Souji  ROKUSHIKA (2) G. Stein and J. Weiss, J. Chem. Soc., 53, 3256 (1949). (3) E. S. G. Barron, J. Ambrose and P. Johnson, Rad. Res., 2, 145 (1955). (4) C. R. Maxwell, D. C. Peterson and N. E. Sharpless, Rad. Res., 1, 530 (1945). (5) C. R. Maxwell, D. C. Peterson and W. C. White, Rad. Res., 2, 431 (1955). (6) B. M. Weeks and W. M. Garrison, Rad. Res., 9, 291 (1958). (7) W. M. Garrison, M. E. Jayko and W. Bennett, Rad. Res., 16, 483 (1962). (8) H. Hatano, J. Rad. Res., 1, 23 (1960). (9) H. Hatano, J. Rad. Res., 1, 28 (1960). (10) H. Hatano, J. Sad. Res., 1, 38 (1960). (11) H. Hatano, This Bulletin, 39, 120 (1961). (12) H. Katsuki, unpublished. (13) S. Shimizu, S. Tanaka and Y. Nakayama, This Bulletin, 37, 306 (1959). ( 82 )

Oxidative Deamination of Several Amino Acids in Aqueous Solution by Gamma Irradiation (Special Issue on Physical, Chemical and Biological Effects of Gamma Radiation, IV)

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Oxidative Deamination of Several Amino Acids in Aqueous Solution by Gamma Irradiation (Special Issue on Physical, Chemical and Biological Effects of Gamma Radiation, IV)

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