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Phosphoenolpyruvate Carboxylase - Assay

CAS:
9067-77-0
EC:
4.1.1.31

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