 Alkaline phosphatase

Alkaline phosphatase, EC3.1.3.1, is a phosphatase activity that hydrolyzes a phosphate monoester to an alcohol and inorganic phosphate. “Alkaline” refers to the activity's alkaline pH optimum under suitable assay conditions; it does not specify one universal optimum for every enzyme preparation or substrate. The accepted activity has broad substrate specificity.^[ref]

A useful optical assay follows hydrolysis of 4-Nitrophenyl phosphate. The resulting 4-Nitrophenol is present mainly in its colored anionic form in the alkaline measurement mixture, so increasing absorbance reports product formation. The manufacturer's method measures the change at405nm and subtracts an enzyme-free blank.^[ref]

Using the Beer-Lambert law, $A=\epsilon \ell c$, a constant optical path and calibrated coefficient giveFor an illustrative blank-corrected slope $0.185\mathrm{mi}{\mathrm{n}}^{-1}$, path length $1\mathrm{cm}$ and the method's coefficient $18.5\mathrm{m}{\mathrm{M}}^{-1}\mathrm{c}{\mathrm{m}}^{-1}$, product appears at $0.010\mathrm{mM}\mathrm{mi}{\mathrm{n}}^{-1}$, or10micromolar per minute. The coefficient is assay-dependent, not a universal constant for every pH and wavelength.^[ref]

This assay measures catalytic activity under its stated conditions. Inferring enzyme amount requires additional calibration and assumptions about activity per molecule; a colored signal alone does not count protein molecules.