Colorimetric Estimation of Inorganic Phosphate and Acid Phosphatase Activity in Plant Tissue Homogenates *

Colorimetric procedures for the determination of phosphate utilizing the stable yellow color which is developed when an excess of molybdate is added to an acidified solution of orthophosphate and vanadate have been described.2'8 Other workers have developed methods whereby inorganic phosphate and the activity of phosphatases may be estimated in blood serum.1"'5 Although cytochemical techniques for the study of the distribution of phosphatases in tissues have been greatly refined in recent years, methods for the quantitative estimation of the activities of these enzymes in plant homogenates or natural fluids are needed. The method described here was developed during investigations now in progress in this laboratory on the distribution of enzymes in the developing embryo sac of Cocos nucifera. These procedures have been extensively used with coconut milk and endosperm homogenates,6 and preliminary tests indicate that they may be satisfactorily applied to the estimation of inorganic phosphate and acid phosphatase activity in other plant materials. The method measures the amount of residual inorganic phosphate present in homogenates; after incubation of these homogenates with a suitable substrate under controlled conditions, inorganic phosphate is again determined. Phosphatase activity is calculated from the difference between total inorganic phosphate after incubation with substrate and the residual inorganic phospfate of the homogenate.