Synthetic peptides and fluorogenic substrates related to the reactive sitesequence of Kunitz-type inhibitors isolated from Bauhinia: Interaction with human plasma kallikrein

We have previously described Kunitz-type serine proteinase inhibitors purif ied from Bauhinia seeds. Human plasma kallikrein shows different susceptibi lity to those inhibitors. In this communication, we describe the interactio n of human plasma kallikrein with fluorogenic and non-fluorogenic peptides based on the Bauhinia inhibitors' reactive site. The hydrolysis of the subs trate based on the a. variegata inhibitor reactive site sequence, Abz-WISAL PRSVFIQ-EDDnp (K-m 1.42 muM, k(cat) 0.06 s(-1), and k(cat)/K-m 4.23 x 10(4) M-1 s(-1)), is more favorable than that of Abz-VMIAALPRTMFIQ-EDDnp, relate d to the B. ungulata sequence (K-m 0.43 muM, k(cat) 0.00017 s(-1), and k(ca t)/K-m 3.9 x 10(2) M-1 s(-1)). Human plasma kallikrein does not hydrolyze t he substrates Abz-RPGLPVRFESPL-EDDnp and Abz-FESPLRINIIKE-EDDnp based on th e a. bauhinioides inhibitor reactive site sequence, the most effective inhi bitor of the enzyme. These peptides are competitive inhibitors with K-i val ues in the nM range. The synthetic peptide containing 19 amino acids based on the a. bauhinioides inhibitor reactive site (RPGLPVRFESPL) is poorly cle aved by kallikrein. The given substrates are highly specific for trypsin an d chymotrypsin hydrolysis. Other serine proteinases such as factor Xa, fact or XII, thrombin and plasmin do not hydrolyze a. bauhinioides inhibitor rel ated substrates.