Nα benzoyl l arginine 4 nitroanilide hydrochloride bapna

The inhibitory activity of Cip1 against papain (Sigma-Aldrich) was determined by assaying the proteolytic activity of 30 μl of 1 mg/ml papain in Tris-HCl buffer, pH 6.8 in the presence of 1 mM glutathione, using 1.5 mM Nα-Benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPNA, Sigma-Aldrich) as the substrate in the presence or absence of Cip1. The kinetic parameters for substrate hydrolysis were determined by measuring the initial rate of enzymatic activity. The inhibition constant Ki was determined with the Lineweaver-Burk equation. The Ki value was also calculated from the double reciprocal equation by fitting the data into the computer software Origin 6.1. For the Lineweaver-Burk analysis, 1 μM papain was incubated with and without 3.98 nM and 7.46 nM inhibitor and assayed at increasing concentration of BAPNA (0.1–5 mM) at 37°C for 30 min. The reciprocals of substrate hydrolysis (1/V) for inhibitor concentration were plotted against the reciprocal of the substrate concentration, and the Ki was determined by fitting the resulting data. For comparison of Cip1 with mutant Cip1, 4.1 μM papain was incubated with 0.56 μM (mutant) Cip1 inhibitor or chicken cystatin and 0.2 mM BAPNA in a 100 μl volume. Substrate conversion was monitored by increased fluorescence at 410 nm over time using an Infinite M200 Tecan microtiter plate reader.