Peptides hydrolysis, assay

Assays performed at 25 °C, pH 7.5,1.0 M NaCt and a buffer concentration of 0.05MTris for peptide hydrolysis and 10 4MTris for ester hydrolysis. ... 

Enzyme regulation and interpretation of activity assays Little to no information is available on the regulation of these enzymes although extracellular peptide hydrolysis and amino acid oxidation appear to be sensitive to the nutrient environment (MulhoUand et ai, 2004a,b MuIhoUand and Lee, in revision). 

Figure 10, Phenyl acetate inhibition of carboxypeptidase A-catalyzed hydrolysis of peptide, Dns-(Gly)g-i.-Phe, 1 X and the ester, Dns-(Gly)3-L.-0Phe, 4 X iO M (57), Enzyme concentrations were 5 X iO" and 4 X IO" M for ester and peptide hydrolysis, respectively (48, 57). Assays were performed in the absence (dashed Une) and presence (solid line) of 1 X phenyl acetate, 0Ac, at 25°C and...

Figure 10. Inhibitory effects of peptides from chymotryptic hydrolysates of modified bovine serum albumin (BSA) on hydrolysis of BSA by bovine a-chy-motrypsin. BSA was acetylated and then reductively methylated. Eight ml (0.87 mg/ml) of this product in 0.012M Tris buffer and 0.012M CaCl2, pH 7.7, was incubated at 37°C for 24 hr with 200 fig of a-chymotrypsin. The solution was then boiled for 10 min. Aliquots of this mixture of peptides were then added in the indicated proportions of BSA to the peptides. The various ratios of BSA to peptides in assay were 17 1 (%) 3.5 1 (M) 1.7 1 ( ). BSA alone (O). The BSA concentration was 0.2 mg/ml in 0.02M borate buffer at pH 8.2 ana the a-chymotrypsin was 3.2 fig/ml. The hydrolysis was followed according to a modification of the procedure by Lin et al. (62) [Figure from Ref. 56].

The versatility of the reagent system in the assay of small peptides is nicely illustrated in Figures 3 through 5. Neurotensin, a polypeptide composed of 13 amino acids, and three fragments from partial hydrolysis were derivatized with NDA/CN and separated by... 

The hydrolysis of peptide bonds catalyzed by the serine proteases has been the reaction most extensively studied by low-temperature trapping experiments. The reasons for this preference are the ease of availability of substrates and purified enzymes, the stability of the proteins to extremes of pH, temperature, and organic solvent, and the existence of a well-characterized covalent acyl-enzyme intermediate. Both amides and esters are substrates for the serine proteases, and a number of chromo-phoric substrates have been synthesized to simplify assay by spectrophotometric techniques. 

In one case, a small peptide with enzyme-like capability has been claimed. On the basis of model building and conformation studies, the peptide Glu-Phe-Ala-Ala-Glu-Glu-Phe-Ala-Ser-Phe was synthesized in the hope that the carboxyl groups in the center of the model would act like the carboxyl groups in lysozyme 17). The kinetic data in this article come from assays of cell wall lysis of M. lysodeikticus, chitin hydrolysis, and dextran hydrolysis. All of these assays are turbidimetric. Although details of the assay procedures were not given, the final equilibrium positions are apparently different for the reaction catalyzed by lysozyme and the reaction catalyzed by the decapeptide. Similar peptide models for proteases were made on the basis of empirical rules for predicting polypeptide conformations. These materials had no amidase activity and esterase activity only slightly better than that of histidine 59, 60). 

Figure 5. Separation of ozonized lysozyme CNBr fragments. The ozonized lysozyme was treated with CNBr as described in the Materials and Methods. The peptide mixture was applied to a column of Sephadex G-25 (3 X 100 cm) and eluted with 0.2N HAc at a flow rate of 0.5 ml/min. Fractions were assayed by measuring UV absorbance on ninhydrin color after alkaline hydrolysis.

The International Union of Biochemistry and Molecular Biology recommends that the term peptidase be used synonymously with the term peptide hydrolase (IUBMB, 1992). Thus, in this unit the term peptidase is used in reference to any enzyme that catalyzes the hydrolysis of peptide bonds, without distinguishing between exo- and endopeptidase activities. Peptidases may be assayed using native or modified proteins, peptides, or synthetic substrates. In this unit, the focus is on assays based on the hydrolysis of common, commercially available, protein substrates. Thus, the assays are not intended to be selective for a given peptidase they are designed to provide estimates of overall peptidase activity. Other units in this publication focus on synthetic or model substrates, which can be designed for the measurement of specific endo- and/or exopeptidase activities. 

Enzyme activities are based on rates of casein hydrolysis under defined conditions. The products of casein hydrolysis, as defined in this protocol, are those peptides soluble in 5% TCA that can be detected by the bicinchoninic acid (BCA) protein assay (unitbi.i). The amount of TCA-soluble peptide generated during the course of the reaction can actually be quantified by any one of several protein/peptide assays. The color yield in these assays is assumed to be proportional to the amount of peptide in solution. The amount of product/peptide in the reaction mixture is often reported as bovine serum albumin (BSA) equivalents—since standard curves based on this protein may be used to calibrate the assay. Thus, activity units can be expressed as the amount of BSA equivalents generated per unit time. 

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