Pyruvate Kinase Assay

Method: The reaction velocity is determined in a lactate dehydrogenase coupled assay system by measuring the decrease in absorbance at 340 nm resulting from the oxidation of NADH.

One unit of activity causes the oxidation of one micromole of NADH per minute at 25°C and pH 7.6 under the specified conditions.

Reagents

• 0.05 M Imidazole⋅HCl buffer, pH 7.6, containing 0.12 M potassium chloride and 0.062 M magnesium sulfate
• 45 mM Adenosine diphosphate
• 45 mM Phosphoenolpyruvate
• 6.6 mM NADH
• Lactate dehydrogenase (Code: BHLDHC). Dilute lactate dehydrogenase to a concentration of 1300-1400 units/ml in above imidazole buffer. Keep cold during use.

Enzyme

Dilute immediately before use to obtain a rate of 0.02-0.04 ΔA/minute.

The protein concentration of a solution of the purified enzyme may be determined as follows:

Procedure

Adjust spectrophotometer to 340 nm and 25°C.

Pipette into cuvettes as follows:

0.05 M Imidazole⋅HCl buffer, pH 7.6	2.7 ml
45 mM Adenosine diphosphate	0.1 ml
6.6 mM NADH	0.1 ml
45 mM Phosphoenolpyruvate	0.1 ml
Lactate dehydrogenase	0.01 ml

Mix well and incubate in spectrophotometer for 4-5 minutes to achieve temperature equilibrium and establish blank rate, if any. Add 0.01 ml of diluted enzyme and record decrease in A₃₄₀ for 4-5 minutes. Calculate ΔA₃₄₀/minute from the initial linear portion of the curve.

Note: Initial absorbance at 340 nm should be 1.4 ±0.1. If not, the NADH may be impure or improperly prepared and should not be used.

Calculation