A high performance liquid chromatographic method for the determination of kinin releasing activity of kallikrein

Abstract

An HPLC method for measuring the in vitro kinin-forming activity of Kallikrein (K), is described. Kinins are formed by incubating K with partially purified bovine kininogen. An aliquot portion of the incubation solution is injected directly onto the chromatographic column (RP-18, 10 um, length 25 cm, 4 mm i.d.). Kallidin and bradykinin are separated from each other and ballast substances by gradient elution technique (phosphate buffer, acetonitrile) and column switching. By post column derivatization kinins are reacted with the fluorogenic reagent Fluram and quantified online, using a fluorescence detector. Detection limits for kallidin and bradykinin are approximately 3 ng = 2.5 pico mol. The reproducibility of the results is 2% (relative standard deviation). This method has been used to study the kinin-forming activity of different K formulations and the results were compared with those obtained by bioassay. As a main result the reaction kinetics demonstrate that purified K solely produces kallidin. Bradykinin showing up later in the reaction mixture must be due to impurities of kininogen which convert kallidin to bradykinin. K itself does not convert kallidin to bradykinin.