Two Kinins Formed by Trypsin from Human Plasma Protein

Abstract

Trypsin was found to generate depressor substances from human plasma protein fraction IV-4 at all pH values from 2.0-10.0. The depressor substances generated at three representative pH values, i.e., 3.2, 5.0, and 8.0 were purified and isolated. The procedures used for the isolation were: (a) gel-filtration through Sephadex G-25; (b) desalting by ion retardation chromatography on AG 11 A 8; (c) equilibrium chromatography on SP-Sephadex C-25; (d) desalting by AG 11 A 8 again, and (e) partition chromatography on a high pressure liquid chromatograph. Upon high pressure liquid chromatography, the depressor substances formed at each of the three pH values were separated into two fractions. In thin layer electrophoresis, the two depressor substances moved with mobilities identical with those of synthetic bradykinin and synthetic [des-Pro³]-bradykinin but not those of methionyl-lysyl- or lysyl-bradykinin. The amino acid analysis of the hydrolysate of the depressor substance 1 generated at pH 3.2 showed the following proportional composition: Arg, 2; Pro, 2; Gly, 1; Phe, 2; Ser, 1. The product thus lacked 1 mol of proline residue as compared with bradykinin, and the retention time of this depressor substance 1 was identical with that of synthetic [des-Pro³]-bradykinin on high pressure liquid chromatography. The depressor substance 2 showed Arg, 2; Pro, 3; Gly, 1; Phe, 2; Ser, 1. This is identical with the composition of bradykinin. Finally, substances 1 and 2 showed oxytocic activities identical to those of synthetic [des-Pro³]-bradykinin and synthetic bradykinin, respectively. Substance 1 showed a bradykinin-potentiating activity similar to that of synthetic [des-Pro³]-bradykinin.