Colorimetric determination of orthophosphate in the assay of inorganic pyrophosphatase activity

Using Baginski and Zak's (12) method for determining inorganic orthophosphate as a starting point, a number of conditions which influence the accuracy and precision of the determination of pyrophosphatase activity have been shown: nonenzyme-catalyzed acid hydrolysis by protein precipitation agents and molybdate-catalyzed hydrolysis of pyrophosphate together with interference in the determination of orthophosphate by the substrate pyrophosphate and other components from the reaction mixture, Tris, chloride, acetate, citrate, boric acid. With regard to these sources of error, a method is described for determining pyrophosphatase activity, and its reliability is investigated.     

Measurement of inorganic orthophosphate in biological materials: Extraction properties of butyl acetate

The properties of several organic solvents were investigated to determine their suitability for use in phosphomolybdic acid (PMA) extraction procedures for the measurement of inorganic orthophosphate (P_i). Butyl acetate exhibited the most satisfactory properties for measurements at 310 nm, which is the absorption peak for unreduced PMA. Butyl acetate exhibits essentially no absorption at 310 nm, is highly selective for PMA over molybdate and silicomolybdate, does not extract molybdate in the presence of trichloroacetic acid (TCA), exhibits negligible volume changes when equilibrated with equal volumes of aqueous phase, and is among the least toxic of organic solvents. Optimal conditions of acidity, molybdate concentration, and reaction and extraction times were determined for the formation of PMA and extraction into butyl acetate. A procedure for measurement of P_i in biological material employing 8% TCA for precipitation of organic material at 0°C, reaction of the supernatant with acid molybdate, extraction of the PMA with butyl acetate and reading of unreduced PMA at 310 nm is described. The procedure is simple, rapid, accurate, selective, and has high sensitivity. Because of the short (20 sec) sample exposure to acid molybdate, it is suggested that the procedure may also be useful in the measurement of P_i in the presence of adenosine triphosphate such as for the assay of adenosinetriphosphatase activity.     

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

A new and convenient method for the determination of P_i was developed. Phosphomolybdate is measured colorimetrically, without reduction to molybdenum blue, by dissolving the whole assay mixture in acetone, where phosphomolybdate is bright yellow. The hydrolysis of acid-labile 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 H₂O₂. 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 P_i. The application of the method to the assay of inorganic pyrophosphatase in the cells of Escherichia coli is described.     

Analytical determination of orthophosphate in water

Methods for orthophosphate determination and the problems of interferences are reviewed.An important group of methods utilizes the phosphomolybdate complex. The complexation step, the reduction step and the extraction step are treated separately and alternative procedures compared.Another group of methods uses ion association complexes; they are primarily used in physiology and not commonly used in water analyses today.Enzymatic methods for orthophosphate analysis in natural waters have been developed lately and are ready for application in selected waterbodies.Flame spectroscopic, fluorometric, gas chromatographic, ion exclusion chromatographic, inductively coupled plasma and other methods are also shortly presented.Radiobiological bioassays for orthophosphate are also available.In conclusion it was emphasized that the most common and reliable technique still is the molybdenum blue method as modified by Murphy & Riley (1962).The need for more specific and sensitive methods is particularly strong in investigations of phosphorus turnover and phosphorus limitation in natural waters. For these purposes the enzymatic phosphatase methods has advantages due to their specificity for orthophosphate and they might offer an alternative to the molybdenum blue method.     

Linear one-step assay for the determination of orthophosphate

A rapid one-step spectrophotometric assay for orthophosphate that requires a single stable reagent solution is presented. The reagent solution, an aqueous mixture of ammonium molybdate and zinc acetate at pH 5.0, produces a stable complex with orthophosphate that absorbs strongly in the near-visible region of the light spectrum. Response to concentration of phosphate was linear up to 300 microM phosphate with a molar absorptivity of 7200 M-1 cm-1 at 350 nm. The mild conditions for phosphate determination employed in this method are unique, making it particularly suitable for the assay of orthophosphate in the presence of labile organophosphates.     

The determination of aminoketones in biological fluids

• 1.1. A new chromatographic procedure is described for the determination of aminoacetone and δ-aminolevulinic acid (after conversion to pyrroles) in both tissues and urine following their separation from porphobilinogen.
• 2.2. The method for measurement of both porphobilinogen and aminoketones involves only two chromatographic procedures, both utilizing Dowex-1 acetate resin. It permits concentration of pyrroles derived from both aminoketones, providing for the determination of as little as 20-mμmole quantities of these substances. Reproducibility and recoveries are excellent over a wide range of concentrations.
• 3.3. Aminoacetone pyrrole is retained by the resin primarily by adsorption; δ-aminolevulinic acid pyrrole is bound by an ionic mechanism.
• 4.4. Aminoacetone constitutes a significant portion of the aminoketones in nonporphyric urines.
• 5.5. The administration of relatively large doses of threonine results in an increased urinary excretion of aminoacetone.