Application of hydrophobic interaction chromatography for purification of pig urinary kallikrein. Characterization of the enzyme and its antibody.

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 2 500- to 3 000-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 molecular weight of 47 000. Isoelectric focusing in flat bed polyacrylamide gels revealed one stained band and one 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.