Stopped-flow time-tracesofFRET-peptidehydrolysis by HNE.
A, Stopped-flow fluorescence kinetic recording of 3.8 µMFRET-peptide hydrolysis by 12.6 nM HNEat 25°C in 10 mM Tris-HCl buffer, pH 7.4, containing 100 mM NaCl. The progress of the reaction was monitored by the fluorescence increase of the released product recorded on 2 adjacent time regions with distinct sampling periods: 0.5 ms from 0 to 2 s, 2 ms from 2 to 6 s. Gray solid line represents the best fit obtained from the mechanism depicted in Scheme I in the absence of heparin with the aid of DynaFit IV® software (see Experimental Procedures). The insert graphic represents the associate residual errors from the best fit curve with experimental data. B, the HNE species as a function of time reaction: free enzyme, E (–); complex enzyme-substrate, ES (– •–) and acyl-enzyme, ES' (- - -).