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.

formulas

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

calculation

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