Lawrence Berkeley National Laboratory Lawrence Berkeley National Laboratory Title THE RADIATION CHEMISTRY OP AMINO ACIDS, PEPTIDES AND PROTEINS IN RELATION TO THE RADIATION STERILIZATION OF HIGH-PROTEIN FOODS Permalink https://escholarship.org/uc/item/3f9466pm Author Garrison, W.M. Publication Date 1979-03-01 Peer reviewed eScholarship.org Powered by the California Digital Library University of California LBL-8928 Warren M. Garrison ABSTRACT An important source of information on :he question of whether or not toxic or other deleterious substances are formed in the radiation sterilization of foods Is the chemical study of reaction products and reaction mechanisms in the radiolysis of individual food components. The present evaluation of the radiation chemistry of amino acids, peptides and proteins outlit.es the various radiation- induced processes which lead to amino acid degradation and to the synthesis of amino acid derivatives of higher molecular weight. Among the latter are the a.a'-diaraino dicarboxylic adds which are formed as major products in the radiolysis of peptides both in aqueous solution and in the solid state. The a,a1-diamino acids are of particular interest as irradiation products because they represent a class of compounds not normally encountered in plant and animal protein sources. Such compounds have, however, been isolated from certain types of bacteria and pathogenic toxins. All of the available data strongly suggest that the a,a'-diamino acids are produced in significant yield in the radiation sterilization of high protein foods. The Importance of initiating extensive chemical and bio­ logical studies of these and of other high molecular weight products in irradiated food is emphasized. NOTICE Thia report wu prepared u in account of work aponsored by dw United States Government. Nettie* the United Statei not the United Slates Department of Energy, nor my of their employees, nor any of their conlraciora, eubcontractars, or their employee!, makes any warranty, exprela or implied, or assumes any legal liability or retponslbilily f.-.r the accuracy, compteteneu at usefulness af any tnfohnaiion, apparatus, product cr process disclosed, or rep,*aenu thai lis use would not Infringe privately owned lights. ttmMi&uiuw w tm uooumwT is u«u«msi»i 1 THE RADIATION CHEMISTRY OF AMINO ACIDS, PEPTIDES AND PROTEINS IN RELATION TO THE RADIATION STERILIZATION OF HIGH-PROTEIN FOODS Warren M. Garrisont Materials and Molecular Research Division Lawrence Berkeley Laboratory, University of California Berkeley, California 94720 1. Introduction The use of ionizing radiation for the preservation of foods offers extraordinary possibilities for greatly increasing the availability of foodstuffs throughout the world. Broad economic and social advantages would be derived from the development of a successful food irradiation technology. In recent years it has been shown that the radiation sterilization of meats in the frozen state in the absence of oxygen yields products with essentially the same taste, aroma and color as the unirradiated samples. ' The question of the wholesomeness of irradiated high-protein foods is receiving careful consideration. Extensive biological testing of the nutritional and toxicological aspects of the holesomeness problem are in 1-4 progress in a number of countries. Chemical identification of products formed in the radiolysis of food constituents offers an important potential source of information on the question of whether or not toxic or other deleterious compounds are formed. Major chemical components of meat include water, protein and lipid in the approximate percentages of 65, 25 and 15 percent respectively. Present address: P. 0. Box 744, Alamo CA 94507 2 Radiation chemical change in the protein and lipid fractions from energy absorbed directly, in the organic component and from the in­ direct action of reactive radical species formed in the radiation decom­ position of water. We review here the radiation chemistry of amino acids, peptides and protein in aqueous solution and in the solid state with particular reference to the subject of product identification. Although the concentration of free amino acids in biological tissue is relatively low, we include a discussion of their radiation chem­ istry because such studies have provided basic information in the develop­ ment of our understanding of the more complex radiation chemistry of pep­ tides and protein. 2. Amino Acids in Aqueous Solution The radiolysis of water is well described. ' The formation of major decomposition products can be summarized in terms of the formula- tier.. H„0— > H,0,,H-,OH,H,eH_0«.,H„,OH.H.e'" .,H + (1) i. i i. aq where s represents the hydrated electron. For y-rays and fast electrons the 100 eV yields, (G), of the free radical products correspond to G(OH) = 2.8, G(e" ) = 2.7 G(H) = 0.55, The reactions of the major radical products, of e and OH, with the simpler a-amino acids, glycine and alanine in oxygen-free solution 8—12 yields ammonia, keto acid and fatty acid as major products. Detailed chemical studies of chese systems, including the use of added second solutes for the preferential scavenging of e and OH, led to formulation of the reaction scheme 3 e~ + NH^CH(R)COO~ •* NH3 + CH(R)COO~ (2) OH + NH^CIURJCOO" -*• H20 + NH^C(R)COO" (3) followed by _ CH(R)C00~ + NHuCH(R)COO~ + CH2(R)COO~ + NHtc(R)COO (4) _ CH(R)COO~ + NH^C(R)COO" ->• CH2(R)COO + NH2=C(R)COO~ (5) - 2 NH^C (R) COO" -•• NH2=C (R) COO" + MltcH (R) COO (6 ) The labile imino acid derivative produced in the disproportionation steps 5,6 hydrolyzes spontaneously H20 + NH2=C(R)COO" -v NH* + RCOCOO~ (7) The overall stoichiometry of reactions 2-7 gives GCPH-) ^G(RCOCOOH) + G (C^RCOOH) ~ 5 The yield of higher molecular-weight products from glycine and +• - alanine is low. Radicals of the type NH_C(R)COO disproportionate almost quantitatively as shown in steps 5,6. In the case of glycine a small +• - fraction of the NH.CHCOO radicals undergoes dimerization to yield 12 a,a'-diamino8uccinic acid COOH- CH2(NH2) - CH2(NH2) - COOH Since neither ethylamine nor (3-alanine were found to undergo the reductive deamination reaction 2, it was proposed that e adds to the C=0 bond of the a-amino acid and that the reduced intermediate then dissociates. 4 e + NHTCH(R)COO •*• NBLCH(R)C-/ -> NH. + CH(R)COO (2a) aq 3 3 \Q 3 The radical products of reactions 2,3 have since been studied quite ex­ tensively by the pulse radiolysis technique. The reaction sequence 2a has also been observed in ESR studies of the reactions of photo-generated 18 electrons with amino acid in aqueous glasses at low temperatures. With the aliphatic amino acids of higher molecular weight, i.e., with ct-amlnobutyric acid, valine, leucine etc. The reductive examination reaction 2(2a) continues to represent a major path for removal of e- 19-22 However, with the longer aliphatic side-chains, the analogues of reactions 3,4 are no longer confined to the C-H bond at the a-carbon position. Other sites along the chain become involved. The radicals so formed react as typical aliphatic carbon radicals and preferentially dimerize rather than D disproportionate via reactions 5b. With a-aminobutyric acid, for example, both a,a'-diamine suberic acid ° ^ COOH-CH(NH„)-(CH-) -CH(NH„)-COOH„ 1 l L l »® and a,a'-diaminomethyl pimelic acid COOH-CH(NH2)-(CH2) -CH(CH3)-CH(NH2)-COOH are formed as major products with a combined yield of G ~3. 19 Studies of product yields in the radiolysis of phenylalanine in neutral oxygen-free solution show that a major fraction of e is removed via the reductive deamination reaction 2. ' Recent pulse radiolysis studies indicate that ~50 percent of a reacts via 2 while the remainder aq 25 adds to the aromatic ring. The OH radical reacts both at the a-carbon oo 26 27 position via 3 and also through ring addition. ' ' Comparison of the 5 observed yields for phenylalanine destruction, G(-Ph) with the yields of phenylpropionic acid, phenylpyruvic and tyrosine indicate that unidenti­ fied higher molecular-weight products are produced In appreciable yield 23 to account for G(-Ph) ~5. By analogy with results obtained in radioly- 28 29 sis studies of aqueous benzene, ' one of these (dimer) products would correspond to the a,a'-diamino acid. COOH-CH (NH2) -CH2-/~~ V-/~A -CH,,-CH (NH2) --COOH Both e and OH react with tryptophan, and histidine almost quan- 30-33 titatively through ring addition. However, chemical studies show that the net destruction of solute is considerably less than G(0H)+G(e~ ) aq 31 ~ 5. For example with tryptophan G(-M) < 1 in oxygen-free solution. The evidence is that in the radiolysis of unsaturated ring systems a recon- stitution reaction is involved" ' ' OH + M -»• MOH (8) e~ + M -»• MH + 0H~ (9) aq MOH + MH -> M + M(HOH) (10) M(HOH) •*• M + H20 (11) The presence of a second solute at concentrations sufficient to preferentially scavenge OH in these systems leads to an enhancement in the yield for solute destruction since the possibility for self protection 35 through water elimination (reaction 10) is precluded i.e. 6 OH + RH •*• R (12) MH + R •+ MH(R) (13) The radiation chemistry of cysteine (NH*CH(CH2SH)C00~) and other aliphatic thiols in dilute oxygen free solution occurs exiusively at the ou 36j37 SH group e" + RSH •> R + HS~(H„S) (14) aq 2 OH + RSH •*• RS + H2) (15) followed by R + RSH -> RH + RS (16) 2RS -> RSSR (17) to give G(cystine) — G(alanine) + GOH-S) ~ 3. Pulse radiolysis studies 38 39 are in accord with the above formulation. ' Similar chemistry has been observed with penicillamine. The dimer product cystine (RSSR) in equation (17) is of course, an a,a'-diamino acid.