Studies on plasma inhibitors of plasma kallikrfin using chromogenic peptide substrate assays

Abstract Inhibition of plasma kallikrein (KK) by human plasma was studied utilising a chromogenic peptide substrate for this enzyme (Chromozyme-PK, Pentapharm, Basel, Switzerland). Inhibition was found to be both “immediate” and “progressive” and complete inhibition of the enzyme did not occur even after prolonged incubation with plasma. When plasma fractions obtained by Sephadex G-150 gel filtration were tested for “immediate” inhibition three inhibitor peaks were obtained. The plasma components responsible for this inhibitor were identified as α 2 -macroglobulin (α 2 M), C 1 -esterase inhibitor (CIINH) and a protein with a molecular weight similar to α 1 -antitrypsin (α 1 AT). When fractions were tested for “total” inhibition (incubation of fraction with enzyme for 300 seconds at 37°C) CIINH was found to be the major inhibitor. Both the α 2 M and low molecular weight inhibitor-containing fractions exhibited “progressive” inhibition. Studies with purified antiprotease preparations confirmed that both CIINH and α 2 M exhibit “immediate” and “progressive” inhibition of KK. Preparations of α 1 AT, antithrombin III and α 2 -antiplasmin gave little or no inhibition of KK. The third inhibitor of KK is therefore probably distinct from these antiproteases and has been named kallikrein inhibitor 3 (KKI 3). When “total” kallikrein inhibition in plasma samples from 20 normal subjects was compared with concentrations of CIINH, α 2 M and α 1 AT a good correlation was found between CIINH concentration and percentage inhibition. These results suggest that inhibition of plasma kallikrein by plasma is a slow process and indicate that CIINH is the major plasma inhibitor of this enzyme.