Phenolphthalein Monophosphate as a Substrate for the Determination of Alkaline Phosphatase Activity in Tissue Homogenates

Abstract

A simple and accurate method is described for the quantitative determination of alkaline phosphatase in tissue homogenates, utilizing stable buffered phenolphthalein monophosphate as substrate. The compound, which is hydrolyzed at a rate linear to enzyme concentration and incubation time, provides a chromogen directly for spectrophotometric determination. The assay does not require the addition of a non-specific protein or Mg++ for optimal activity and is equally reactive in glass and plastic flasks. The technique affords good sensitivity by measuring enzyme activity in homogenates as dilute as 0.0005% (small intestine). Quadruplicate determinations varied <1%. Small intestine was the most reactive of the rat organs tested, followed by the kidney, lung, heart and spleen.