Abstract
Publisher Summary This chapter presents the procedure of purification and assay of human plasma carboxypeptidase N. Two-step chromatographic procedure for purifying carboxypeptidase N from outdated pooled human plasma is described. It consists of ion-exchange chromatography on a column of DE-52 cellulose followed by affinity chromatography on a column of p-aminobenzoyl-L-arginine-Sepharose- 6B. Enzyme purified 2600-fold was eluted from the resin by the competitive inhibitor guanidinoethylmercaptosuccinic acid. Carboxypeptidase N has both peptidasC; and esterase activity and can be assayed conveniently in the spectrophotometer by either activity. Recent studies have shown a pronounced enhancement of peptidase activity with a penultimate alanine, which may explain the more rapid inactivation of C3a than of C5a by this enzyme. Additional assays are sensitive multistep procedures using hydrolysis of protamine or extraction of benzoyl (Bz)-Gly from Bz-Gly-Lys reaction mixtures H or biological assays.
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