Application of Hydrophobie Interaction Chromatography for Purification of Pig Urinary Kallikrein. Characterization of the Enzyme and its Antibody

Publisher Website
Google Scholar
Abstract
Pig urinary kallikrein was purified following its kinin-generating activity as well as its esterase activity on N.alpha.-benzoyl-L-arginine ethyl ether (BzArgOEt). The isolation procedure, based on hydrophobic interaction chromatography on octyl-Sepharose and affinity chromatography on aprotinin-Sepharose resulted in a 2500- to 3000-fold purified enzyme with a yield of 6%. The preparation was homogenous on alkaline polyacrylamide disc gel electrophoresis and exhibited a single polypeptide chain in dodecyl sulfate-polyacrylamide gel electrophoresis upon reduction and alkylation corresponding to a MW of 47,000. Isoelectric focusing in flat bed polyacrylamide gels revealed 1 stained band and 1 coinciding symmetrical peak of activities which could be eluted between pH 3.6 and pH 4.1 from a replica gel. Monospecific antibodies could be raised in a rabbit and kinetic studies for kinin-generating activity and esterolytic activity (substrate BzArgOEt) of the enzyme as well as for antibody-induced inhibition of the enzyme were performed. The antibody inhibited the antigen via a competitive mechanism near or at the active site of the enzyme.

This publication has 19 references indexed in Scilit: