A new and convenient colorimetric determination of inorganic orthophosphate and its application to the assay of inorganic pyrophosphatase

✍ Scribed by Jukka K. Heinonen; Reijo J. Lahti

• Publisher: Elsevier Science
• Year: 1981
• Tongue: English
• Weight: 408 KB
• Volume: 113
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(81)90082-8

✦ Synopsis

A new and convenient method for the determination of P, was developed. Phosphomolybdate is measured calorimetrically, without reduction to molybdenum blue, by dissolving the whole assay mixture in acetone, where phosphomolybdate is bright yellow. The hydrolysis of acidlabile phosphates (e.g., creatine phosphate) causes no problems, because extra molybdate is complexed with citrate immediately after the color has been developed. Strong reductants and SH compounds which interfere, if present in high concentrations, are eliminated by adding H102. Detergents, organic bases, protein, and sucrose do not interfere. The assay is as sensitive as most modifications of the Fiske-SubbaRow method. In the routine procedure the useful range is 50-1500 nmol of Pi. The application of the method to the assay of inorganic pyrophosphatase in the cells of Escherichia coli is described.

' Abbreviations used: Phase, inorganic pyrophosphatase (EC 3.6.1.1); AAM solution, acetone/5 N sulfuric acid/IO mM ammonium heptamolybdate 2/l/l; KU, Klett unit. One Klett unit corresponds to the absorbance of 0.005.