Time-course measurement of enzymatic activity corrected for impacts of disturbances relating to the reaction of the enzyme with a substrate

A method of determination of the course of an enzyme activity that is variable in time, wherein said activity is probed by conversion of a substrate of the enzyme, comprising the steps of:

• a) in a selected test set up and for a determined substrate of the enzyme, determining the velocity of signal production (dFdiag/dt) resulting from a time curve of the signal (Fdiao=f(A)) obtained from splitting said substrate when it is contacted with a determined initially fixed concentration of the enzyme (E) and providing a “diagnostic plot” with the values of (dFdiag/dt) against the signal (Fdiag) and determining whether said diagnostic plot is either a straight line or a parabola;
• either over a part of—or over the entire plot, and determining the value vinit which is the intercept of the diagnostic plot with the ordinate and a which is its intercept with the abscissa.
• b) in the same test conditions, for a given test sample, determining the signal production (Fexp) resulting from splitting the substrate as in a) by the enzyme generating in and/or disappearing from the sample and providing the time curve of signal Fexp=f(t);
• c) transforming the obtained experimental value of the signal (Fexp) in step b) into an ideal value (Ftransf) by applying the following transformation:
• (i) If and for so far as the diagnostic plot is a straight line, Ftransf=−(αln (1−Fexp/α);
• (ii) if and for so far as the diagnostic plot is a parabolic line, Ftransf=αarctanh (Fexp/α).
• d) determining the enzyme concentration (Eexp), over the time from the transformed signal values (Ftransf)) as Eexp=VinitFtransf/dt.