Products of radiolysis of tyrosine and glycyl-L-tyrosine in oxygen-free nitrogen, N2O or air saturated water solution, pH 4.0 or 8.6 were analysed in an amino acid analyser and upon separation on DEAE cellulose or BioGel P-2 column with spectrofluorimetry. Apart from dihydroxyphenylalanine tyrosine solution irradiated in nitrogen or N2O contained dityrosine, while that of irradiated glycyl-L-tyrosine contained glycyl-dityrosine-glycine. Both dimeric products were formed with radiation yields, of about 0.15 at low pH value. In addition an unknown, nonfluorescent but ninhydrin positive product was found in irradiated tyrosine solution. Another, unidentified product of radiolysis was detected in N2O or air saturated solutions of tyrosine. The product had a blue-green fluorescence and a molecular weight of about 500.
Insulin, ribonuclease, papain and collagen solutions saturated with nitrogen, N2O or air were irradiated with doses of 10 to 640 Gy of gamma rays. Protein solutions were also oxidized enzymatically in a system of horse-radish peroxidase: hydrogen peroxide. Column chromatography (Sephadex G-75 or Sephacryl S-200) of treated protein solutions revealed that they contain protein molecular aggregates. Nitrogen saturation of solution before irradiation was most favourable for radiation-induced aggregation of proteins. Fluorescence analysis of protein solutions resulted in detection of dityrosyl structures in irradiated as well as in enzymatically oxidized proteins. Concentration of dityrosine in proteins studied was determined fluorimetrically in their hydrolysates separated on BioGel P-2 column. In irradiated proteins, dityrosine was present almost exclusively in their aggregated forms. In proteins oxidized enzymatically, dityrosine was also present in fractions containing apparently unchanged protein. Mechanisms which could account for differences in the yield of dityrosine formation in radiolysis and in enzymatic oxidation of proteins are suggested.
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