Instructions For UseReagents:
0.05 M Sodium pyrophosphate buffer, pH 8.8.
96% Ethanol (substrate).
0.025 M NAD+ (16.7 mg/ml) in buffer. Prepare fresh.
0.01 M Sodium phosphate buffer, pH 7.5, containing 0.1% bovine serum albumin (BSA).
Alcohol dehydrogenase (enzyme) - Dissolve sufficient amount of enzyme in 0.01 M sodium phosphate buffer containing 0.1% BSA, pH 7.5, to give a concentration of 0.1-0.5 U/ml. Prepare fresh immediately prior to assay.Procedure:
1. Set spectrophotometer (equipped with strip chart recorder and temperature control) at 340 nm and 25°C.
2. In a cuvette pipette the following reagents in the amounts indicated:
|Sodium pyrophosphate buffer|| 1.4 ml|
|NAD+ || 1.4 ml|
|Ethanol (substrate)|| 0.1 ml|
3. Incubate cuvette in spectrophotometer, at 25°C for 5 min. to achieve temperature equilibration and then record absorbance at 340 nm (blank).
4. Initiate the reaction by adding 0.1 ml of ADH (enzyme) solution to the cuvette. Record the increase in absorbance at 340 nm for 5 min.
5. Calculate the E340nm/min