Determination of inorganic phosphate in the presence of triton X-100

✍ Scribed by Yew Phew See; P.S. Fitt

• Publisher: Elsevier Science
• Year: 1972
• Tongue: English
• Weight: 304 KB
• Volume: 49
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(72)90445-9

✦ Synopsis

During our studies of the polynucleotide phosphorylase of rat liver mitochondrial membranes (1,2), we have needed to assay phosphatase, pyrophosphatase, and nucleoside diphosphatase activities in the presence of Triton X-100 used (2) to solubilize the membrane-bound polynucleotide phosphorylase. The assays depended on the determination of orthophosphate and it is known (3,4), and we have confirmed, that Triton X-100 interferes with the Fiske and SubbaRow method (5,6) by precipitating the phosphomolybdate complex. Eibl and Lands (4) found that the increase in turbidity in the absence of a reducing agent was proportional to orthophosphate concentration and could be made the basis of a sensitive phosphate assay, but their method was not convenient for our purposes owing to its low upper limit of proportionality.

Roufogalis (7) has recently published a modification of the Fiske and Subba-Row assay that can be used in the presence of Triton X-100 and certain other interfering substances, which are removed by prior precipitation with silicotungstic acid.

We have found that the precipitated phosphomolybdate complex redissolves if the concentration of Triton X-100 is sufficiently high and we have made use of this observation to develop two rapid methods for the accurate estimation of orthophosphate in the presence of the detergent. They are modifications of the Gomori modification of the Fiske and SubbaRow procedure (5,6) and of the Lowry-Lopez method (8,9), respectively. MATERIALS Triton X-100 was purchased from Canadian Laboratory Supplies Ltd., Montreal, P.Q., and p-methylaminophenol sulfate (Elon) from Eastman Kodak Co., Rochester, N. Y. All other reagents were obtained from Fisher Scientific Co., Ottawa, Ont.