Peptidase Activity Assays Using Protein Substrates

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. 

In this unit, Basic Protocol 1 presents a procedure using casein as substrate. The Alternate Protocol describes the modification of this procedure for use with a denatured hemoglobin substrate. Basic Protocol 2 presents a procedure using a chromaphore-conjugated casein derivative, azocasein. For quantitation, the authors have chosen to use either the BCA-based colorimetric assay unitbli) for soluble protein/peptides (in Basic Protocol 1) or the intrinsic absorbance of the chromaphore-conjugated peptide products (in Basic Protocol 2). 

Peptidase Activity Assays Using Protein Substrates 

DETERMINATION OF PEPTIDASE ACTIVITY USING A CASEIN SUBSTRATE 

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. 

---

C2.1 Activity Measurements of Proteinases Using Synthetic Substrates C2.2 Peptidase Activity Assays Using Protein Substrates... 

Peptidase Activity Assays Using Protein Substrates... 

The assays presented in this section deal with the measurement of enzyme activity, which is expected to be proportional to the amount of active enzyme present in a sample, food or otherwise, unit ci.i is an overview of the important considerations in performing activity assays unitc 1.2 illustrates how these considerations are applied to the assay of a representative food-relevant glycosyl hydrolase. Chapters C2, C3, and C4 present the first units on particular types of activity assays. In Chapter C2, two units present peptidase activity assays that use either synthetic substrates (UNITC2.1) or common, commercially available protein substrates (unit C2.2). unitC3.i presents three different assays for lipase activity. unitc4.i presents assays for diphenol oxidases, and unitc4.2 for lipoxygenase. 

---