Reliability of enzymatic localization in tissue sections by the simultaneous coupling techniques is dependent to a great extent upon the speed of coupling. Therefore, the influence on coupling rate of the structure of the diazonium ion and of the coupling component was studied. Electro-negative groups in the diazonium ion increase the rate of coupling, while the same groups in the coupling component decrease the rate. Electro-positive groups in the coupling component accelerate coupling, but slow it when present in the diazonium ion. The relation of coupling rate and electro-negativity of the substituents in the diazonium ion follows Hammett's equation (8). Although the relations is linear on coupling with aromatic hydroxy compounds, it is not so with aromatic amines. The most active diazonium ions showed no increase in coupling rate with aromatic amines. This suggests that for those enzymes hydrolyzing an ester link, increase of coupling rate might be accomplished by modifying the structure of either the coupling component used in the substrate or the diazonium salt. However, for enzymes splitting amide linkages, the only possibility of improving the localization is by modifying the structure of the coupling component.
INFLUENCE OF CHEMICAL STRUCTURE ON THE RATE OF AZO COUPLING AND ITS SIGNIFICANCE IN HISTOCHEMICAL METHODOLOGY