The activity of adenosine deaminase was assayed spectrophotometrically
by measuring the decrease in absorbance at 265 nm on
conversion of adenosine to inosine with a Shimazu spectrophotometer.
One milliliter reactions containing 0.04–0.08 lmole of
adenosine in 50 mM potassium phosphate were incubated for
1 min in a cuvette prior to the addition of enzyme. The amount
of product formed was calculated using a molar extinction coefficient
of 8.4 mM1 [12]. The same wavelength and molar extinction
coefficients were used for determination of the Km and Vmax for
ara-A, 20-deoxyadenosine, adenine, AMP, ADP, and ATP. One unit
of enzyme activity is defined as the amount of enzyme needed to
convert 1 lmole of substrate to product at 37 C in one minute.
Protein concentration was determined by the Bradford method,
using bovine serum albumin as standard.