The pH dependence of spectral parameters for Kalckar's adenosine deaminase assay

Optimal monitor wavelengths and differential millimolar extinction coefficients (m delta epsilon) for rate determination of reactions catalyzed by adenosine deaminases on several substrates have been investigated as a function of pH in the range from 6.5 to 12. The values found are in some cases at variance with those quoted in the biochemical literature. The effect of pH on m delta epsilon values is shown to be clearly related to acid-base properties of product and/or substrate in the reaction. Experimental data are in most cases used to derive analytical functions describing the pH dependence of m delta epsilon. For the conversion of adenosine to inosine at pH 6.5, the following values of m delta epsilon +/- SE were obtained: at 263 nm, 8.27 +/- 0.02; at 264 nm, 8.36 +/- 0.02; at 265 nm, 8.27 +/- 0.03. These represent absolute maximal values as a function of pH.