# Also in the Article

Measurement of inhibition constants

Procedure

Inhibition constants (Ki) were determined by measuring the residual activities of protease in the presence of different dilutions of inhibitor (twofold dilutions) using fluorogenic substrates. Activities were measured at 25°C in buffer containing 10 mM tris-Cl (pH 7.4), 150 mM NaCl, 10 mM MgCl2, 1 mM CaCl2, 0.1% (w/v) BSA, and 0.01% (v/v) Triton X-100 by monitoring the change of fluorescence intensity (excitation, 368 nm; emission, 467 nm) or absorption (405 nm) for 30 min using a Tecan Infinite M200 Pro plate reader. Proteases were of human origin and were purchased from Molecular Innovations or Innovative Research. KLK5 was expressed recombinantly as described elsewhere. The following protease concentrations were used: 0.1 nM trypsin, 2 nM thrombin, 5 nM KLK5, 0.6 nM protein C, 7.5 nM tPA (tissue plasminogen activator), 1.5 nM uPA (urokinase plasminogen activator), 0.25 nM plasma kallikrein, 1 nM factor XIa, 2 nM factor XIIa, and 2.5 nM plasmin. The following fluorogenic substrates were used at a final concentration of 50 μM: Z-Gly-Gly-Arg-AMC for trypsin, thrombin, tPA and uPA, Z-Phe-Arg-AMC for plasma kallikrein, Boc-Phe-Ser-Arg-AMC for factor XIa, Boc-Gln-Gly-Arg-AMC for factor XIIa, and H-D-Val-Leu-Lys-AMC for plasmin. The fluorogenic substrate Val-Pro-Arg-AMC for KLK5 was used at a final concentration of 100 μM. The chromogenic substrate Pyr-Pro-Arg-pNA was used at a final concentration of 0.48 mM to measure protein C activity. The median inhibitory concentration (IC50) values were determined by fitting sigmoidal curves to the data using the following four-parameter logistic equation used for dose response curves$y=1001+10(logIC50−x)p$x is the logarithm of the concentration of the peptide, y is the percentage activity relative to the reaction without peptide, and IC50 values were derived from the fitted curve. p is the Hill coefficient. The Ki values were calculated on the basis of the IC50s using the Cheng-Prusoff equation. The Km for trypsin and thrombin for the substrate Z-Gly-Gly-Arg-AMC were determined to be 90 and 168 μM, respectively, and the Km of KLK5 for Val-Pro-Arg-AMC was 216.3 μM.

Note: The content above has been extracted from a research article, so it may not display correctly.

Q&A