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Stopped-flow time-tracesofFRET-peptidehydrolysis by HNE.

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posted on 20.02.2013, 17:16 by Gabriel L. C. Nunes, Alyne Simões, Fábio H. Dyszy, Claudio S. Shida, Maria A. Juliano, Luiz Juliano, Tarsis F. Gesteira, Helena B. Nader, Gillian Murphy, Alain F. Chaffotte, Michel E. Goldberg, Ivarne L. S. Tersariol, Paulo C. Almeida

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' (- - -).