The isolation of liver serine proteinase by affinity chromatography on 4-phenylbutylamine-sepharose 4 B.

By acid extraction, ethanol precipitation, affinity chromatography on 4-phenylbutylamine-Sepharose 4B and gel filtration on Sephadex G-100, calf liver neutral proteinase was purified. The purified enzyme was electrophoretically homogeneous and over 2000 times more active than the starting homogenate. The molecular weight, determined by SDS electrophoresis, was calculated as 27000. The pH optimum of the enzyme for whole calf thymus histones and N-benzoyltyrosine, ethyl ester (BTEE) was at 7.0 and 7.0-7.5. The Km value for histones was 2% and for BTEE 1.66 mM. The enzyme was strongly inhibited by soya-bean trypsin inhibitor and leucocyte intracellular I-1A inhibitor and less by alpha 1-antitrypsin and leucocyte inhibitor I-1B. The enzyme hydrolyzed only selected protein substrates, such as total thymus histones, Lys-rich histones, nucleoprotein and substance P, but not Arg-rich histones, hemoglobin and casein. The enzyme showed chymotrypsin-like properties by cleavage of substance P at the carboxyl groups of phenylalanine and leucine.