Making enzymatic methods optimum for measuring compounds with a kinetic analyzer.

Kinetic enzymatic methods for analysis of substrates can be made optimum for a sensitive photometric analyzer by adjusting the activity of the triggering (catalyzing) enzyme so that the reaction rate is maximum at the time of measurement. tat this optimum activity, the exponential time constant for exhaustion of substrate equals the time between triggering and rate measurement. The scale factor (defined as measured activity divided by sample concentration in the reaction mixture) is the same for all tests. Sensitivity to substrate concentration is predictable from instrumental absorbance uncertainty and molar absorptivity of the absorbing species. These predictions from Michaelis theory were verified experimentally for pyruvate and lactate triggered with lactate dehydrogenase, for glucose triggered with hexokinase, and for triglycerides triggered with glycerol kinase, the reaction rate being measured 30 s after triggering. Sensitivities of 1.5 times 10(-7) mol/liter were achieved. Serum diluted 1000-fold and analyzed for glucose gave a repeatability of 25 mg/liter with linearity to 4.0 g/liter. Samples diluted 300-fold and analyzed for triglycerides gave 30 mg/liter repeatability, with linearity to concentrations exceeding 3.0 g/liter.