The use of hexazonium-p-rosanilin in the histochemical demonstration of peptidases

Abstract

The suitability of hexazonium-p-rosanilin (HP) in the histochemical demonstration of peptidases was investigated. The detection was carried out in cold mictrotome sections adherent to slides or semipermeable membranes. Alanyl-1-naphthylamide, alanyl-2-naphthylamide, leucyl-2-naphthylamide, leucyl-4-methoxy-2-naphthylamide (all substrates in concentration of 0.4 mg/1 ml of citrate phosphate buffer pH 6.5), gamma-L-glutamyl-1-naphthylamide, gamma-L-glutamyl-2-naphthylamide (both substances in concentration of 0.24 mg/1 ml of acetate buffer pH 6.5) were used as the substrates. Results were compared with those obtained with Fast Blue B and Fast Garnet GBC. In comparison with Fast Blue B and Fast Garnet GBC HP is a faster coupler, furnishes azodyes which are stable, amorphous (even without lipid extractions from sections), more substantive and in the case of 1-naphthylamine almost insoluble in ordinary lipid solvents used for the dehydration and clearing of sections before mounting. The molecular extinction coefficient of azodyes furnished by HP is 1.5X higher for 1-naphthylamine than for 2-naphthylamine. It is higher than that of Fast Garnet GBC, however, lower than that of Fast Blue B. The inhibitory influence of individual diazonium salts on enzyme activity (activities) splitting leucyl-2-naphthylamide amounts to 36% (Fast Garnet GBC), 37% (Fast Blue B), 52% (HP, 0.03 ml/1 ml) and 63% (HP, 0.09 ml/1 ml) at pH 6.5. For gamma-glutamyl-transpeptidase the corresponding values are 50%, 59%, 62% and 67%. The higher inhibitory influence of HP is compensated by the possibility of its using in the technic of semipermeable membranes. HP improves greatly the localization of peptidases in cold microtome sections from which lipids were not extracted. The best results are furnished by 1-naphthylamine dervatives. In the case of 4-methoxy-2-naphthylamine derivatives the localization is very sharp, however, the azodye is less distinct than that of 2-naphthylamine. The localization as obtained with HP in combination with substrates derived of simple naphthylamines is similar or even better than with 4-methoxy-2-naphthylamine derivatives applied with Fast Blue B. Typical examples are shown.