RADIATION-INDUCED PRODUCTS OF PEPTIDES AND THEIR ENZYMATIC DIGESTIBILITY

Chemical characterization of radiation-induced products of peptides and proteins is essential for understanding the effect of ionizing radiation on peptides and proteins. Furthermore, peptides containing radiation-altered amino acid residues might not be completely digestible by proteolytic enzymes. In this work, small homopeptides of Ala, Phe and Met were chosen as model peptides. Lysozyme was used to investigate the effect of ionizing radiation on a small protein. All peptides and lysozyme were irradiated in diluted, oxygen free, N(,2)O-saturated aqueous solutions, using a ('60)Co-(gamma)-source. HPLC, capillary GC and GC-MS were applied to isolate and characterize the radiation-induced products. The enzymatic digestibility of the products was investigated using aminopeptidase M, leucine aminopeptidase, carboxypeptidase A and carboxypeptidase Y. For L-Ala peptides, radiation-induced D-Ala was observed. Biphenyl type dimers and products containing o-, m- and p- Tyr were formed in irradiated solutions of L-Phe peptides. 2-aminobutanoic acid, methionine sulfoxide, 2-amino-4-(methyldithio)butanoic acid, 4,4'-thiobis(2-aminobutanoic acid) and homocystine were identified as radiation-induced products of Met peptides after HCl-hydrolysis. Radiation-induced D-Met was also observed. For irradiated lysozyme, allo-Thr, 2-aminobutanoic acid, o- and m-Tyr and 2- and 3-hydroxytyrosines were identified after HCl-hydrolysis. Yields of radiation-induced products in all cases were also determined by GC. Enzymatic hydrolysis of irradiated Ala, Phe and Met homopeptides demonstrated the different actions of four enzymes used on radiation-induced products. Peptide bonds of radiation-induced dimers of di-Ala were resistant to enzymatic hydrolysis whereas those of radiation-induced dimers of Phe-peptides were readily cleaved by the enzymes except for carboxypeptidase Y. For Met-peptides, peptide bonds of radiation-induced products were cleaved by all four exopeptidases in most cases. In conclusion, it was found that irradiation of peptides examined in this work leads to racemization and alteration of amino acid residues and crosslinks between the peptide chains. In addition, it was established that exopeptidases act differently on radiation-induced dimers of peptides composed of aliphatic, aromatic and sulfur-containing amino acids.