Malate Dehydrogenase Assay
Method: The reaction velocity is determined by measuring the decrease in absorbance at 340 nm resulting from the oxidation of NADH. One unit oxidizes one micromole of NADH per minute at 25°C and pH 7.4 under the specified conditions.
Note: NADH may vary in salt form and degree of hydration. Care should be exercised to use an analytical grade and to use the correct molecular weight.
Immediately before use, dilute in 0.1 M phosphate buffer, pH 7.4 to obtain a rate of 0.02-0.04 ΔA/minute.
Set spectrophotometer at 25°C and 340 nm. Pipette into each cuvette as follows:
Incubate cuvettes in spectrophotometer for 3-4 minutes to achieve temperature equilibration and establish blank rate, if any. Add 0.1 ml diluted enzyme to cuvette and record decrease in ΔA340 for 3-5 minutes. Calculate ΔA340 per minute from the initial linear portion of the curve.