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.

Reagents

• 0.1 M Potassium phosphate buffer, pH 7.4
• 0.006 M Oxaloacetic acid, freshly prepared in 0.1 M phosphate buffer pH 7.4. This reagent is unstable and should be stored in an ice bath during use.
• 0.00375 M NADH, freshly prepared in 0.1 M potassium phosphate buffer, pH 7.4

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.

Enzyme

Immediately before use, dilute in 0.1 M phosphate buffer, pH 7.4 to obtain a rate of 0.02-0.04 ΔA/minute.

Procedure

Set spectrophotometer at 25°C and 340 nm. Pipette into each cuvette as follows:

0.1 M Phosphate buffer	2.6 ml
NADH	0.2 ml
Oxaloacetate	0.1 ml

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.

Calculation