(38) Assay Card

General Information
Summary Chymotrypsin and trypsin Ka values determined for SFTI-1 analogues.
Condition Increasing amounts of inhibitor were added to a constant amount of enzyme, and, after suitable incubation time, the residual enzyme activity was measured on a Cary 3E spectrophotometer (Varian, Australia) by using a turnover substrate. Enzyme┬Činhibitor interactions were determined in Tris-HCl buffer (0.1m, pH 8.3) containing CaCl2 (20 mm) and 0.005% Triton X-100 at 228C. The measurements were carried out at initial enzyme concentrations of 5.3 nm and 6 nm for trypsin and chymotrypsin, respectively. After proper incubation time, the residual enzyme activity was measured with Phe-Val-Pro-Arg-Anb5,2-NH2 for analogue 1 and Suc-Ala-Ala-Pro-Leu-4-nitroanilide for analogue 2. The experimental points were analysed based on the plot of [enzyme] versus [Io].
Result Reported Ka values for chymotrypsin and trypsin for N-SFTI-1-C of 9.9x10^10 M^-1 and 4.9x10^6 M^-1 respectively, for [Nlys5]SFTI-1 of 1.0x10^8 M^-1 against trypsin and for [Nphe5]SFTI-1 3.8x10^8 M^-1 against chymotrypsin.
AssayType Protease inhibition

Stawikowski M, Stawikowska R, Jaskiewicz A, Zablotna E, Rolka K (2005) Examples of Peptide-Peptoid Hybrid Serine Protease Inhibitors Based on the Trypsin Inhibitor SFTI-1 with Complete Protease Resistance at the P1-P1' Reactive Site. Chembiochem 6:1057-61

Proteins Assayed N-SFTI-1-C