A new linear plot for standard curves in kinetic substrate assays extended above the Michaelis-Menten constant: application to a luminometric assay of glycerol.

In conventional kinetic substrate assays the standard curve is plotted as observed reaction rate, upsilon obs, versus added substrate concentration, Sadd, and has a linearity limited to Sadd much less than Km. From this plot the blank reaction rate, upsilon bl, is easily estimated but not the contaminating substrate concentration, Scon, present in reagents (unless it is the only blank source). Thus the actual substrate concentration, S = Scon + Sadd, cannot be estimated as required for the various linear plots based on the Michaelis-Menten equation. We have derived an expression, (upsilon obs - upsilon bl)/Vapp = Sadd/(Kmapp + Sadd), containing only those parameters measured for a conventional standard curve (Vapp and Kmapp are obtained from a plot of (upsilon obs - upsilon bl) versus (upsilon obs - upsilon bl)/Sadd). A plot of (upsilon obs - upsilon bl)/Vapp versus Sadd/(Kmapp + Sadd) can be used as a standard curve with the following advantages over the conventional standard curve: (a) For all kinetic substrate assays it is identical and connects the points (0, 0) and (1, 1). Thus deviations from true Michaelis-Menten kinetics or erroneous kinetic constants are easily detected. (b) Since it is linear even above Km, the analytically useful range is considerably extended. (c) For assays with a wide dynamic range it can be used in lin-lin or log-log form. The procedure is illustrated for a kinetic assay of glycerol (Kmapp = 40 mumol/liter). The plot was found to be entirely linear in the range 0.07-100 mumol/liter (glycerol concentration in cuvette).