The correction of reaction rates in continuous fluorometric assays of enzymes.
The kinetic data obtained from the action of a cathepsin D-like enzyme from Biomphalaria glabrata hepatopancreas (digestive gland) on MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNp)-D-Arg-NH(2), was studied as a data prototype, generated by means of a fluorogenic substrate. An initial fluorescence, due to incomplete energy transfer, of about 8% of the values attained after complete substrate hydrolysis; a non-linear standard curve even at microM concentrations and an exponential decay of the steady state fluorescence of reaction product of the order of 10(-4) x s(-1) were the main analytical problems encountered. The standard curves for fluorescence of the substrate reaction product after 48 h of hydrolysis, and the reference compound MOCAc-Pro-Leu-Gly-NH(2), were fitted by polynomial approximation and the point derivates used as calibration factors. Time dependence of the calibration factor for the reaction product was -2.96 x 10(-4) a.u microM(-1) x s(-1) that is, in the same order of observed enzymic reaction rates. A mathematical treatment was devised for obtaining rates corrected for errors derived from the three analytical problems indicated. The method is of general application in continuous fluorometric assays, irrespective of the particular enzyme used, but of special value for substrates that present significant initial fluorescence. The reaction rates were 11% higher; as calculated by means of the calibration factor [substrate]/(final-initial fluorescence intensities), which is the prevalent procedure in the literature; leading to underestimation of K(m) and overestimation of V(max).
Alves
Antônio Carlos Vassalo
AC
Department of Physiology and Biophysics, Institute of Biological Sciences (UFMG), Avenida Pres. Antônio Carlos 6627, CEP 31270-901-Campus Pampulha, Belo Horizonte, MG Brazil.
Rogana
Edyr
E
Barbosa
Célia de Fátima
Cde F
Ferreira-Alves
Dalton L
DL
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000032:analysis / Q000378:metabolism; D001700:Biomphalaria / Q000201:enzymology; D002402:Cathepsin D / Q000032:analysis / Q000378:metabolism; D004798:Enzymes / Q000032:analysis* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods* / Q000706:statistics & numerical data; D005456:Fluorescent Dyes; D043143:Hepatopancreas / Q000201:enzymology; D007700:Kinetics; D009842:Oligopeptides / Q000737:chemistry; D013379:Substrate Specificity
D004798:Enzymes; D005456:Fluorescent Dyes; D009842:Oligopeptides; D016282:Aspartic Acid Endopeptidases; D002402:Cathepsin D