STUDIES ON STRUCTURES OF POLAR DYES DERIVED FROM LIVER PROTEINS OF RATS FED N-METHYL-4-AMINOAZOBENZENE .3. TYROSINE AND HOMOCYSTEINE SULFOXIDE POLAR DYES

The protein-bound dyes formed in the livers of rats fed the hepatocarcinogen N-methyl-4-aminoazobenzene were hydrolyzed to give polar dyes separable by chromatography on silica and thin layers of cellulose. The polar dyes Pla, Plb, and Pic were characterized in this study. The synthetic ester A-benzoyloxy-N- methyl-4-aminoazobenzene reacted at pH 7 with tyrosine to form two polar dyes which were identical with Pla and Plb in all respects tested (electronic spectra in acetate buffer and 6 n HCl; thin-layer chromatography on cellulose in a basic, an acidic, and two neutral solvent systems before and after methylation of the dyes; and tests with ninhydrin, nitrous acid, and phenol reagent). Through a variety of reactions the tyrosine polar dyes were provisionally characterized as Ar-(3-tyrosyl)-Ar-methyl-4-aminoazobenzene (Pla) and 3-(3-tyrosyl)-V-methyl-4-aminoazobenzene (Plb). Syn thetic 3-(homocystein-S-yl)-7V-methyl-4-aminoazobenzene, previously proved to be identical with the major polar dye P2b, was easily oxidized by H2O2 to a sulfoxide that was identical with the minor polar dye Pic. The four dyes P2b, Pla, Plb, and Pic accounted for 90% of the polar dyes obtained by the successive enzymatic and hot alkaline hydrolysis of the hepatic protein-bound dyes. Neither the polar dye derived from the reaction of tryptophan with N-benzoyloxy-N-methyl-4-aminoazobenzene nor radioactivity was detected among the polar dyes derived from the liver proteins of rats fed Ar-methyl-4-aminoazobenzene and tryptophan-2′,3′-3H. N-Benzoyloxy-N-methyl-4-aminoazobenzene reacted with cysteine to form a polar dye that was destroyed completely by the hot alkaline hydrolysis used in the isolation of the polar dyes from the liver proteins. © 1969, American Chemical Society. All rights reserved.