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Abstract
A standard assay for measuring alkaline phosphatase activity is detecting the production of nitrophenol from the artificial substrate p-nitrophenol phosphate. Nitrophenol absorbs light at 420 nm, which provides a convenient detection method. However, the absorbance of nitrophenol varies with pH. This variation complicates the use of the colorimetric assay to study the pH-dependence of alkaline phosphatase activity. We have determined the standard curve relating the absorbance of nitrophenol to buffer pH. These levels were used to normalize data collected from studies of alkaline phosphatase catalysis of different concentrations of p-nitrophenol phosphate across various reaction pH levels. The studies indicate that alkaline phosphatase activity increases over three-fold from pH 7 to pH 9 at saturating concentrations of p-nitrophenol phosphate. Both the V and K of the enzyme max m increased as the reaction pH was increased. This indicated a possible conformational relaxation of the alkaline phosphatase catalytic site to allow an elevated rate of catalysis. It is also possible that the ionization state of one or more residues in the catalytic site of alkaline phosphatase has been altered by the pH shift. This too could account for the pH-dependence of the enzyme’s V and K. max m