Lactate Dehydrogenase, L- (Cytochrome b2) Assay

Method Essentially that described by Appleby and Morton (1959). The rate of reduction of potassium ferricyanide is determined spectrophotometrically at 420 nm. The molar absorbancy of potassium ferricyanide is 1.04 X 10³ at this wavelength whereas the reduced ferricyanide has negligible absorption. One unit reduces one micromole of ferricyanide per minute at 25°C and pH 8.4 under specified conditions.

Reagents

• 0.067 M Sodium phosphate, pH 7.4 with 0.001 M EDTA
• 0.01 M EDTA
• 0.0083 M Potassium ferricyanide. Caution, read product label for handling instructions.
• 0.1 M Sodium pyrophosphate, pH 8.4
• 0.5 M DL Sodium lactate, pH 7.0

Enzyme

Immediately prior to assay, dilute enzyme in 0.067 M phosphate buffer, pH 7.4 with 0.001 M EDTA to obtain a rate of 0.02-0.04 ΔA/minute.

Procedure

Adjust spectrophotometer 420 nm and 25°C. Pipette into cuvettes: 2.0 ml of 0.1 M sodium pyrophosphate, 0.5 ml of 0.5 M DL sodium lactate, 0.3 ml of 0.01 M EDTA, and 0.1 ml of potassium ferricyanide. Incubate in spectrophotometer for 3-4 mintutes at 25°C to reach temperature equilibrium and establish blank rate if any. Add 0.1 ml of diluted enzyme and record A₄₂₀ for 4-5 minutes. Calculate ΔA₄₂₀/minute from initial linear portion of curve.

Calculation