Kinetic data for the chromogenic substrates available from Chromogenix. Suitable chromogenic substrates are listed for a number of serine proteases, most of them part of the cascade systems in blood. Some of the substrates are cleaved by more than one enzyme although at different rates. The kinetic analyses of the enzymatic cleavage of pNA from the substrates were performed under strictly standardized conditions using the clinical chemistry analyzer Cobas Mira S.
A stable, well-defined temperature is vital for all enzyme kinetic studies and in this study all reactions were performed at 37 °C. A suitable buffer was chosen for each enzyme and the pH value given in the compilation is the value to which it was adjusted at 25°C. Note that the pH value of Tris buffers decreases as the temperature increases, at the rate of approximately 0.1 unit per °C (50 mM Tris-HCl). The kinetics of the reaction was followed spectrophotometrically by measuring the change in absorbance over time, ΔA/min. To ensure the highest precision, ΔA/min was measured at four different substrate concentrations. Insertion of the ΔA/min values into Eq. 18, followed by linear regression gave Km, kcat and Vmax for the reaction.