QUANTIFICATION OF KININOGENS IN PLASMA - A FUNCTIONAL METHOD BASED ONTHE CYSTEINE PROTEINASE-INHIBITOR ACTIVITY

We have previously reported on a microassay based on human kininogens as cysteine proteinase inhibitors (CPIs), which could quantify partial ly purified kininogens from different biological fluids (J Pharmacol M eth 26, 113-124, 1991). In the present study we describe a functional method that, when assuming a 1:1 stoichiometry between papain and kini nogen, allows a direct measurement of the molar concentration of kinin ogens in plasma. The principle of the method is that the target enzyme papain is inhibited by kininogens present in added diluted plasma. Th e residual activity of papain, not inhibited in this reaction, subsequ ently hydrolyzes the added peptide substrate (S-2302), generating a ye llow colour which is read in a microplate reader at 405 nm. Relating t he test samples to a standard curve established from known concentrati ons of E-64 (a selective low molecular weight inhibitor of cysteine pr oteinases), we could quantify kininogens on a molar basis. A major pro blem when first applying this method to plasma, was the interference o f alpha(2)-macroglobulin, which inhibited papain and generated a compl ex able to split the chromogenic substrate. The interference of alpha( 2)-macroglobulin was eliminated by an initial acid treatment of plasma followed by dilution with a buffer containing methylamine. The specif icity for kininogens in this assay is demonstrated by the following ob servations: Commercial pooled normal plasma contained 3.2 mu M CPI act ivity, in good agreement with the expected molar concentration of kini nogens. After gel filtration of a plasma sample with a CPI activity of 3.4 mu M, two peaks with CPI activity were identified as H-kininogen (0.9 mu M) and L-kininogen (2.5 mu M), both in good accordance with ex pected concentrations of the two kininogens. Plasma deficient of kinin ogens had a minimal inhibitory capacity towards papain.