On the validity of continuous spectrophotometric assays for adenosine deaminase activity: a critical reappraisal

Kinetic investigations on adenosine deaminase from calf intestinal mucosa by spectrophotometric monitoring of the reaction at 264, 270, or 228 nm show that this method does not produce artifactual inhibition by substrate excess up to 0.7 mM concentration, when either adenosine or 2'-deoxyadenosine are employed with calf adenosine deaminase. The evaluation of kinetic parameters for this system was carried out both by initial rate measurements and by numerical differentiation of time progress curves according to a recently published method (S. C. Koerber and A. L. Fink, 1987, Anal. Biochem. 165, 75-87). The following results were obtained by the latter method at pH 7.0 and 30 degrees C: for the conversion of adenosine to inosine, kcat = 251 +/- 15 s-1, KMs = 29.7 +/- 2.8 microM, KMp = 613 +/- 62 microM; for the conversion of 2'-deoxyadenosine to 2'-deoxyinosine, kcat = 283 +/- 17 s-1, KMs = 22.4 +/- 2.2 microM, KMp = 331 +/- 35 microM. At 285 nm, a slight negative deviation from Beer's law was observed for adenosine at concentrations higher than 0.9 mM. No deviation was found for inosine up to 2.0 mM at the same wavelength.