Malate Dehydrogenase - Assay
Source:
Porcine Heart Mitochondria
CAS:
9067-93-0
EC:
1.1.1.37
Malate Dehydrogenase Assay
Method
The reaction velocity is determined by measuring the decrease in absorbance at 340 nm resulting from the oxidation of NADH. One unit oxidizes one micromole of NADH per minute at 25°C and pH 7.4 under the specified conditions.
Reagents
- 0.1 M Potassium phosphate buffer, pH 7.4
- 0.006 M Oxaloacetic acid, freshly prepared in 0.1 M phosphate buffer pH 7.4. This reagent is unstable and should be stored in an ice bath during use.
- 0.00375 M NADH, freshly prepared in 0.1 M potassium phosphate buffer, pH 7.4
Note: NADH may vary in salt form and degree of hydration. Care should be exercised to use an analytical grade and to use the correct molecular weight.
Enzyme
Immediately before use, dilute in 0.1 M phosphate buffer, pH 7.4 to obtain a rate of 0.02-0.04 ΔA/minute.
Procedure
Set spectrophotometer at 25°C and 340 nm. Pipette into each cuvette as follows:
0.1 M Phosphate buffer 2.6 ml NADH 0.2 ml Oxaloacetate 0.1 ml
Incubate cuvettes in spectrophotometer for 3-4 minutes to achieve temperature equilibration and establish blank rate, if any. Add 0.1 ml diluted enzyme to cuvette and record decrease in ΔA340 for 3-5 minutes. Calculate ΔA340 per minute from the initial linear portion of the curve.
Calculation
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