An improved method for determination of the total kininogen in rabbit and human plasma

Abstract

The method described by Diniz and Carvalho has been widely used for determination of the total kininogen (KGN) in plasma, but this method is also reported to produce bradykinin (BK) potentiators besides BK after incubation of the pretreated plasma with trypsin. The present experiments aim to establish a method to induce the full conversion of KGN into BK without potentiators and kininase. The pretreatment of plasma with heat (98°) and acetic acid (pH 3.5) used in Diniz's method was compared with five other pretreatments, namely non-treated, two ways of heating (at 98 or 60°) and two ways of acidification (at pH 3.5 or 2.0). The pretreated plasmas were incubated with trypsin in 0.2 M Tris buffer, pH 7.8. BK released was assayed on the isolated rat uterus. The full conversion of KGN into BK by trypsin was tested, comparing it with the amounts of BK released by highly purified hog pancreas kallikrein and highly purified snake venom kininogenase. Trypsin (200 μg) and 30 min of the incubation were sufficient to convert KGN into BK up to plateau level in non-treated and treated plasmas. The results indicated that the pretreatments of Diniz and 98° produced BK potentiators, and the non-treated and the pH 3.5-treated plasmas showed lesser amounts of BK formed, even in the presence of o-phenanthroline. The pretreatments of pH 2.0 and 60° produced no potentiators and caused the full conversion of KGN into BK, but the latter treatment did not inactivate kininase in rabbit plasma. Thus, it is concluded that the pretreatment of plasma with HCl (pH 2.0) was most suitable for the determination of total KGN in rabbit plasma. For human plasma, pretreatment by heating (60°) was also suitable, besides the pH 2.0 treatment.