A rapid one-step assay for intestinal peptide hydrolase activity in mucosal homogenates, and in cytosol and particulate fractions prepared from homogenates is described. Peptide hydrolysis, and estimation of liberated L-amino acids by use of L-amino acid oxidase, peroxidase, and a chromogen are achieved by a single incubation step. The most suitable peptide substrates are dipeptides composed of one amino acid which reacts strongly and rapidly with the L-amino acid oxidase reagent and one which does not react. Brush border peptide hydrolase activity may also be estimated with this assay by performing the incubation in the presence of p-hydroxymercuribenzoate.
A number of methods have been described for the assay of intestinal peptide hydrolase activity. The spectrophotometric assay described by Josefsson and Lindberg (1) is not suitable for the assay of enzyme activity in tissue homogenates or for assay of particulate-bound enzyme. The Matheson and Tattrie method has the limitation of high blank values when some dipeptides are used as substrates (2). Recently, L-amino acid oxidase has been employed to measure the L-amino acids released after peptide hydrolysis (3-5). These methods involve two incubation steps, first, hydrolysis of the substrate by the intestinal enzyme, and secondly after interruption of this reaction, estimation of the released amino acids by incubation with L-amino acid oxidase, horseradish peroxidase and a chromogen. The similarity of these methods to the two-step assay for intestinal disaccharidases (6) and the conversion of this disaccharidase assay to a one-step procedure (7). suggested that a one-step peptide hydrolase assay might be successfully developed.
This paper describes such a method for assay of peptide hydrolase activity in cytosol and particulate fractions of rat intestinal mucosal homogenates and for the determination of brush border peptide hydrolase activity.