title: Standardization of papain reagents by measurement of active sites using a synthetic inhibitor, E-64.
abstract: L-trans-epoxysuccinyl-leucylamido (4-guanidino) butane (E-64) reacts rapidly and irreversibly in a one-to-one ratio with the active site of papain, causing complete inhibition of the enzyme. After the addition of various concentrations of E-64 to a papain preparation, the residual enzyme activity can be measured using an azoprotein technique. The molarity of E-64 required to cause complete inhibition of papain activity is equal to the molarity of papain-active sites. Preparations of papain from various sources were assayed for protease activity by hydrolysis of azoalbumin using several variants of the basic technique and also by hydrolysis of azocasein. For each variant of azoprotein assay procedure, the active sites of the papain were measured using E-64. All variations of the azoprotein technique yielded similar estimates of the active site molarity of the papain preparations, whereas the azoprotein assay results alone showed wide variation. Quantitation of the active-site molarity of various papain preparations using E-64 correlated with serologic efficacy.
authors.affiliation: Blood Group Reference Laboratory, Oxford, United Kingdom.
authors.forename: M L
authors.initials: ML
authors.lastname: Scott
authors.forename: C M
authors.initials: CM
authors.lastname: Whitton
mesh_terms: D001391:Azo Compounds / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D005189:False Positive Reactions; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D010206:Papain / Q000037:antagonists & inhibitors / Q000592:standards*; D011480:Protease Inhibitors / Q000494:pharmacology*
chemical_list: D001391:Azo Compounds; D007202:Indicators and Reagents; D011480:Protease Inhibitors; D010206:Papain; D007930:Leucine; C024974:E 64