Phosphoenolpyruvate Carboxylase Assay

Method: The formation of oxaloacetate is monitored spectrophotometrically in a malate dehydrogenase coupled system. The reaction velocity is measured as a decrease in A₃₄₀ resulting from the oxidation of NADH. One unit oxidizes one micromole of NADH per minute at 25deg.C and pH 8.5 under the specified conditions.

Reagents

• 0.11 M Tris sulfate buffer, pH 8.5
• 0.3 M Magnesium sulfate
• 6.0 mM NADH in 0.11 M Tris sulfate buffer, pH 8.5
• Malate dehydrogenase (WBC Code MACDHCL) dissolved to a concentration of 600 units/ml in 0.11 M Tris sulfate buffer, pH 8.5
• 0.1 M Sodium bicarbonate in reagent grade water
• 0.03 M Phosphoenolpyruvate (PEP), either potassium or cyclohexylammonium salt in 0.11 M Tris sulfate buffer, pH 8.5
• Dioxane: fresh reagent, low in peroxide content
• 0.3 M Dithioerythritol (DTE) in in 0.11 M Tris sulfate buffer, pH 8.5

Enzyme

Dissolve at a mg/ml in 0.11 M Tris sulfate buffer, pH 8.5 containing 0.005 M magnesium sulfate.

Procedure

Adjust the spectrophotometer to 25°C and 340 nm.

Into a cuvette pipette the following:

0.11 M Tris sulfate buffer, pH 8.5	1.8 ml
6.0 mM NADH	0.1 ml
0.3 M Magnesium sulfate	0.1 ml
0.3 M DTE	0.1 ml
0.1 M Sodium bicarbonate	0.3 ml
Dioxane	0.3 ml

Incubate in spectrophotometer for 4-5 minutes to achieve temperature equilibration. Add 0.1 ml of diluted enzyme and 0.01 ml of malate dehydrogenase. Record the decrease in A₃₄₀ to establish blank rate. Add 0.1 ml PEP and record decrease in A₃₄₀ (test rate).

Calculation