Glycerol Dehydrogenase Assay

Method: The activity is measured using the procedure of Lin and Magasanik (1960) by determining the increase in absorbance at 340 nm resulting from the reduction of NAD. One unit reduces one micromole of NAD per minute at 25°C and pH 10.0 under the specified conditions.

Reagents

• 1.0 M Glycerol
• 0.125 M Potassium carbonate
• 0.125 M Sodium bicarbonate
• 0.125 M Carbonate/bicarbonate buffer, pH 10.0. Prepare by mixing potassium carbonate and sodium bicarbonate to reach pH 10.0.
• 1.0 M Ammonium sulfate
• 0.05 M Potassium phosphate, pH 7.6, containing 0.1 M ammonium sulfate and 0.1 mM manganese chloride
• 0.1 M NAD. Note: NAD may vary in salt form and degree of hydration. Care should be exercised to use an analytical grade and the correct molecular weight.

Enzyme

Dissolve enzyme at a concentration of one mg/ml in 0.05 M potassium phosphate buffer solution. Immediately prior to use dilute further in this buffer to obtain a rate of 0.02 - 0.05 ΔA/min.

Procedure

Adjust spectrophotometer to 340 nm and 25°C.

Pipette into each cuvette as follows:

1.0 M Ammonium sulfate	0.1 ml
0.1 M NAD	0.1 ml
0.125 M Carbonate/bicarbonate buffer, pH 10.0	2.4 ml
1.0 M Glycerol	0.3 ml

Incubate in the spectrophotometer for 4 - 5 minutes to achieve temperature equilibration and establish blank rate, if any. At zero time, add 0.1 ml of appropriately diluted enzyme and mix thoroughly. Record increase in A₃₄₀ for 3 - 5 minutes. Determine ΔA₃₄₀/min from initial linear portion of the curve.

Calculation