Stopped-flow time-traces of FRET-peptidehydrolysis by HNE in the presence of heparin.
A, the stopped-Flow fluorescence kinetic recording of 3.8 µMFRET-peptide hydrolysis by 12.6 nM HNEperformed at 25°C in 10 mM Tris-HCl buffer, pH 7.4, containing 100 mM NaCl supplemented with 50 µM heparin. The progress of the reaction was monitored by the fluorescence increase of the released product recorded on two adjacent time regions with distinct sampling periods: 0.5 ms from 0 to 2 s, 2 ms from 2 to 4 s. Gray solid line represents the best fit deduced from the mechanism depicted in Scheme I in the presence of heparin using 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 in function of time reaction in presence of heparin: complex enzyme-heparin, EH (–); complex enzyme-substrate-heparin, ESH (– •–) and complex acyl-enzyme-heparin, ES'H (- - -).