Azo-coupling reactions of protein-bound amino groups in plant tissue by oxidative deamination method.

Abstract

Fresh freehand bean cotyledon sections were killed and fixed in chilled 70% alcohol, hydrolysed with 1% NaOH solution, and washed in a mixture of chloroform-methanol. Suspended the sections in 5% hydrazine hydrochloride solution in pH 6-7 phosphate buffer and passed CO2 slowly to block free aldehyde groups. Washed and deaminated oxidatively with 1% chloramine-T in pH 7.5 phosphate buffer. The protein-bound aldehydes obtained by the above procedure were demonstrated with (a) Schiff's reagent or malachite green (b) arylhydrazines and (c) arylamines. Among arylhydrazines (1) azobenzenephenylhydrazine sulfonic acid, (2) 2,4-dinitrophenylhydrazine and (3) 3-hydroxy-2-naphthoic acid hydrazide were used. Compound (1) gave a purple dye with protein-bound aldehydes. A hydrazone formed after the condensation of the reagent (2) with protein-bound aldehyde, was reduced, diazotized and coupled with H-acid to produce a red-purple dye. After condensation the reagent (3) was coupled with the stable diazotate of 5-nitro-o-anisidine to produce a bright red dye. Among arylamines, H-, Chicago-, J-, and gamma-acids were used. These acids after having condensed their —NH2 groups with aldehydes were coupled with the stable diazotate of 5-nitro-o-anisidine to produce red dyes.