Lactate Dehydrogenase Assay

Method: The reaction velocity is determined by a decrease in absorbance at 340 nm resulting from the oxidation of NADH. One unit causes the oxidation of one micromole of NADH per minute at 25°C and pH 7.3, under the specified conditions.

Reagents

• 0.2 M Tris⋅HCl, pH 7.3
• 6.6 mM NADH in above 0.2 M Tris⋅HCl buffer, pH 7.3
• 30 mM Sodium pyruvate in above 0.2 Tris⋅HCl buffer, pH 7.3

Enzyme

Dissolve at 1 mg/ml in 0.2 M Tris⋅HCl buffer. Dilute enzyme prior to use to obtain a rate of 0.02-0.04 ΔA/min. in Tris buffer and keep cold.

Determination of Protein Concentration:

Procedure

Set spectrophotometer at 340 nm and 25°C.

Pipette into cuvette as follows:

Tris⋅HCl, 0.2 M pH 7.3	2.8 ml
6.6 mM NADH	0.1 ml
30 mM Sodium pyruvate	0.1 ml

Incubate in the spectrophotometer 4-5 minutes to achieve temperature equilibration and establish a blank rate, if any.

Add 0.1 ml of appropriately diluted enzyme and record ΔA₃₄₀/min from initial linear portion.

Calculation