Glycerol Kinase - Assay
EC:
2.7.1.30
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.
Reagents
- Reagent solution: the required amount of solution should be prepared containing:
ATP 8.5 mM NADH 1.22 mM PEP 2.0 mM Lactate Dehydrogenase
(Worthington code: LADCL)15.3 u/ml Pyruvate kinase
(Worthington code: PKL)7.0 u/ml MgSO4⋅7H2O 28.0 mM Reduced Glutathione 26.0 mM pH adjusted to 7.4 - 0.4 M Glycine, pH 8.9 containing 45 mM potassium carbonate
- 0.1 M Triethanolamine⋅HCl buffer, pH 7.4
- 0.1 M Glycerol
Enzyme
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.
Procedure
Adjust spectrophotometer to 340 nm and 25°C.
Pipette into cuvettes as follows:
Carbonate-glycine buffer 2.1 ml Reagent solution 0.7 ml 0.1 M Glycerol 0.1 ml
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.
Calculation