ANALYTICAL BIOCHEMISTRY 85, 86-89 (1978) A Convenient Method for the ATPase Assay DENIS LEBEL, GUY G. POIRIER, AND ADRIEN R. BEAUDOIN Dipartement de biologie et Unit& de Gastroenttrologie, UniversitC de Sherbrooke, Sirerbrooke, Qudbec, Canada JIK 2RI Received November 9, 1976; accepted September 22, 1977 A new method for the determination of inorganic phosphorus released in ATPase assay has been evaluated. The method is based on the reduction of a phosphomolybdate complex by Elon in a copper acetate buffer. In contrast to current methods, there is no interference by ATP with color development. There is also less or no interference by other compounds usually present in ATPase assay media. The method is simple, sensitive, and reproducible. The method of Fiske and Subbarow (1) for the determination of inorganic phosphate (9) is currently used for ATPase assays and many other enzymes involved in phosphorylation processes. The method has serious inconveniences which include interference by ATP with the color development (2,3) and the instability of the color complex. Moreover, the level of sensitivity, the interferences of many substances with the coloration, and the instability of the reducing agent make it an unsatisfactory method for determination of Pi in the assay of ATPase. This method has been modified to allow the determination of inorganic phosphate in the presence of labile phosphate esters (4). Despite the good sensitivity and stability of the color complex that is obtained with these modifications, there remains the considerable interference with color development by ATP and other commonly used compounds in ATPase assay (5). Here, we propose a method which practically eliminates these interferences. The method is sensitive and the color complex is stable. The conditions of the assay minimize hydrolysis of labile phosphates. MATERIALS Tris-ATP and ADP were purchased from Sigma. EIon’ (p-methyl- aminophenol sulfate) was obtained from the Eastman Kodak Company. All other reagents were ACS reagent-grade chemicals. Reagents. The following reagents were used: (A) copper acetate at pH 4.0: 0.25% copper sulfate pentahydrate and 4.6% sodium acetate trihydrate L Eastman Kodak trademark. 0003-2697/78/0851-0086$02.00/O CopyrigM 0 1978 by Academic Press, Inc. AU rights of mpmduction in any form reserved. 86