Purification and partial characterization of two forms of urinary trypsin inhibitor

Crude urinary trypsin inhibitor was obtained by DEAE-cellulose column chromatography from normal fresh urine. By the purification of the crude urinary inhibitor on successive chromatography methods using Sephacryl S-200, DEAE-cellulose, CM-Sepharose CL-6B and Sephadex G-100, we detected two forms of urinary trypsin inhibitor: form I and form II. The specific activity of form I increased approx. 4-fold with a recovery of 60%, as compared to that of crude urinary trypsin inhibitor. N-terminal amino acids of form I and form II were determined to be alanine and valine, respectively. Molecular weights of forms I and II were estimated to be 67000 and 28000 by gel filtration on Sephadex G-100 and to be 43000 and 19000 by SDS-polyacrylamide gel electrophoresis. S-carboxymethylated form I migrated as a single band corresponding to a molecular weight of 59000 in SDS-polyacrylamide gel electrophoresis. From the results of the determination of a single N-terminal amino acid of form I and a single band of S-carboxymethylated form I, it is indicated that it is composed of single polypeptide chain. And the present study suggests that form I is a native form of trypsin inhibitor in normal human urine and form II is a fragmented product from form I in the purification steps.