Adenosine Deaminase Assay

Method: The reaction velocity is measured by a change in absorbance at 265 nm resulting from the deamination of adenosine. One unit converts one micromole of adenosine to inosine per minute at 25°C and pH 7.4 under the specified conditions.

Reagents

• 1.4 mM Adenosine in 0.05 M Phosphate buffer, pH 7.4
• 0.05 M Phosphate buffer, pH 7.4

Enzyme

Dilute immediately before use in ice cold 0.05 M phosphate buffer, pH 7.4 to a concentration of 0.2 - 0.8 units / ml.

Procedure

Adjust spectrophotometer to 265 nm and 25°C.

Pipette into cuvettes as follows:

0.05 M Phosphate buffer, pH 7.4	2.88 ml
1.4 mM Adenosine	0.1 ml

Incubate in spectrophotometer for 3-5 minutes to achieve temperature equilibration and establish blank rate, if any. At zero time add 0.02 ml enzyme solution and mix thoroughly. Record decrease in A₂₆₅ for 2-3 minutes. Use the initial rate to calculate ΔA₂₆₅/min.

Calculation