Glycerol Kinase Assay
Method: The reaction velocity is measured in a coupled system with pyruvate kinase and lactate dehydrogenase. One unit results in the oxidation of one micromole of NADH per minute at 25°C and pH 8.9 under the specified conditions.
Dissolve at a concentration of one mg/ml in 0.1 M TEA buffer, pH 7.4. Immediately prior to use, dilute further in TEA buffer to obtain a rate of 0.02 - 0.04 ΔA/min.
Adjust spectrophotometer to 340 nm and 25°C.
Pipette into cuvettes as follows:
Incubate in spectrophotometer for 3 - 4 minutes to achieve temperature equilibration and establish blank rate, if any.
Add 0.1 ml of appropriately diluted enzyme and record ΔA340 for 6 - 8 minutes. Determine ΔA340/min from the linear portion of the curve. A short lag period may be observed.