Aspartate Aminotransferase Assay

Method

Aspartate aminotransferase may be assayed spectrophotometrically in a coupled reaction with malate dehydrogenase in the presence of NADH (Karmen 1955; Amador and Wacker 1962). One unit oxidizes one micromole of NADH per minute at 25°C and pH 7.4 under the specified conditions.

Reagents

Prepare reagent mixture containing:

L-aspartate 134.0 mM

2-oxoglutarate 6.64 mM

NADH 0.24 mM

Lactate dehydrogenase 5 u/ml

Malate dehydrogenase 1.25 u/ml

Sodium phosphate buffer, pH 7.4 50 mM

Enzyme

Dissolve enzyme at a concentration of one mg/ml in 0.1 M potassium phosphate pH 7.4. Immediately prior to use, dilute further in this buffer to a concentration of 0.05 - 0.25 u/ml.

Procedure

Adjust spectrophotometer to 340 nm and 25°C. Pipette 2.9 ml of the reagent mixture into cuvette and place in spectrophotometer. Incubate for 3 - 4 minutes to reach temperature equilibrium and establish blank rate, if any. At zero time, add 0.1 ml of apropriately diluted enzyme and record the decrease in A₃₄₀ for 4 - 5 minutes. Calculate ΔA₃₄₀/minute from the initial linear portion of the curve.

Calculation

\frac{\text{Units}}{\text{mg}} = \frac{\frac{\Delta \text{A}_{340}}{\text{min}}}{\frac{\text{6.2 x mg enzyme}}{\text{ml reaction mixture}}}

L-aspartate	134.0 mM
2-oxoglutarate	6.64 mM
NADH	0.24 mM
Lactate dehydrogenase	5 u/ml
Malate dehydrogenase	1.25 u/ml
Sodium phosphate buffer, pH 7.4	50 mM

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Aspartate Aminotransferase - Assay

Aspartate Aminotransferase Assay