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

A method of determining amidolytic enzyme activity in a sample over a given period of time based on conversion of a fluorogenic or chromogenic substrate of the enzyme, comprising the steps of:

• measuring a signal (Fdiag) produced from splitting said substrate by enzymatic activity after contact with a determined initially fixed concentration of the enzyme (E) over a time period (t) so as to correctly obtain calibration under initial conditions, and preparing a diagnostic time curve from Fdiag measured over time;
• preparing a diagnostic plot from a first derivative of said diagnostic time curve against Fdiag, said diagnostic plot being a straight line or a parabolic line at least over a portion of said diagnostic plot, wherein the initial rate of substrate conversion (vinit) is where the diagnostic plot intercepts the ordinate and the theoretical upper limit of the signal (α) is where the diagnostic plot intercepts the abscissa, said diagnostic plot providing calibration under initial conditions;
• measuring an experimental signal produced by the sample (Fexp) resulting from splitting the substrate by enzymatic activity from the enzyme, said enzyme activity generating in and/or disappearing from the sample over a period of time, the Fexp and Fdiag being measured under identical conditions;
• preparing an experimental time curve Fexp=f(t) from the Fexp measured over time;
• transforming the Fexp into an ideal value (Ftransf) by an equation according to:
• (i) Ftransf=−(α ln(1−Fexp/α) for a portion of the diagnostic plot that forms a straight line;
• (ii) Ftransf=α arctan h(Fexp/α) for a portion of the diagnostic plot that forms a parabolic line;
• and
• determining the enzyme concentration (Eexp) from Eexp=vinitFtransf/dt so that the enzymatic activity over a given period of time is determined without continuous comparison to an array of values or a calibrator curve simultaneously obtained from a parallel calibration experiment.