Data for Modified Assay of Benzoyl-Arginine p-Nitroanilide-Hydrolysing
Enzymes in Crude Extracts
The resolution of complex mixtures of proteolytic enzymes is greatly simplified by the use of specific, preferably chromogenic, substrates. The assay is simplified further if the chromogenic substrate is converted directly to a coloured product rather than to one requiring, subsequent modification before it can be calorimetrically estimated. BANAl (1,2) and BAPA' (3) are two chromogenic substrates which have found wide application in the assay of trypsin-like enzymes from a variety of sources. They have also been used for the assay of thiol proteases such as some cathepsins (4,5) and the plant proteases papain ( 6)) ficin ( 7)) and bromelain (8). The use of BANA has the disadvantage that the product formed by enzymic hydrolysis requires subsequent modification (1,2,9, 10) before it can be calorimetrically quantitated. However it does have the advantage than it can be used for the direct location of enzyme bands on gels (1,lO). In contrast., BAPA hydrolysis results directly in the release of a coloured product, p-nitroaniline
(3) and thus has many advantages over BANA for routine assays.
In studies on zymogen activation and the intracellular distribution of BAPA-hydrolysing enzymes in insects, the assays were frequently complicated by the presence of turbid solutions which did not clarify on low speed centrifugation (30009 X 10 min) and which interfered with the accurate spectrophotometric estimation of the p-nitroaniline released. This problem could be simply overcome by extracting the acidified reaction mixture with an equal volume of ethyl acetate. The p-nitroaniline is rapidly and completely extracted into the ethyl acetate layer whilst less than 5% of the substrate, BAPA, is extracted under these conditions. The appropriate spectral data required for this simple modification is presented in this paper.
N-benzoyl-m-arginine-p-nitroanilide HCl was obtained from Schwarz-Mann Research Laboratories and p-nitroaniline was obtained from British Drug Houses Ltd. 1V M Solutions of BAPA and p-nitroaniline were used for spectral recordings. The four solvent systems employed were :