Subsite differences between the active centres of papaya peptidase A and papain as revealed by affinity chromatography. Purification of papaya peptidase A by ionic-strength-dependent affinity adsorption on an immobilized peptide inhibitor of papain.
An affinity column consisting of the specific peptide inhibitor of papain, Gly-Gly (O-benzyl)Tyr-Arg, attached to Sepharose was found to bind the active thiol proteinase papaya peptidase A specifically, but only at an ionic strength significantly higher than the one at which papain is bound. When a mixture of active papaya peptidase A and its irreversibly oxidized contaminant was applied to the column, the active enzyme was bound whereas the inactive material was not. The bound enzyme was released by deionized water and found to contain 1 mol of SH group/mol of protein. The different conditions required for the binding of the two enzymes to the immobilized peptide was shown to reflect different ionic-strength-dependences of the affinity of the two enzymes for the peptide in solution. Whereas the affinity of papain for the inhibitor appears to be insensitive to ionic strength over the range studied, that of papaya peptidase A is ionic-strength-dependent and always lower than that of papain. A rate assay is devised for papaya peptidase A with N-benzyloxycarbonylglycine p-nitrophenyl ester as the substrate at pH 5.5. After calibration against an active-site titration the assay yields the thiol-group concentration without interference from inactive contaminants. For the papaya peptidase A-catalysed hydrolysis of N-benzyloxycarbonylglycine p-nitrophenyl ester at pH 5.5 kcat. was found to be 16.7s-1, which is about 3 times the value found for the same reaction catalysed by papain.
D001665:Binding Sites; D002846:Chromatography, Affinity; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D005998:Glycine / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D009842:Oligopeptides / Q000494:pharmacology; D009994:Osmolar Concentration; D010206:Papain / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011480:Protease Inhibitors
D009842:Oligopeptides; D011480:Protease Inhibitors; C034118:N-benzyloxycarbonylglycine 4-nitrophenyl ester; C041863:glycyl-glycyl-tyrosyl(O-benzyl)-arginine; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D010206:Papain; D005998:Glycine