Phosphomolybdate assay

The Folin-Denis assay is used as a procedure for the quantification of total phenolics in plant materials, food, and beverages. Reduction of phosphomolybdic-phosphotungstic acid (Folin-Denis reagent) to a blue-colored complex in an alkaline solution occurs in the presence of phenolic compounds (Folin and Denis 1912). 

A standard Lowry-based protein assay has been adjusted to the special conditions encountered with skin [126], Basically, proteins reduce an alkaline solution of Cu(II)-tartrate to Cu(I) in a concentration-dependent manner. Then, the formation of a blue complex between Folin-Ciocalteau reagent (a solution of complex polymeric ions formed from phosphomolybdic and phosphotungstic heteropoly acids) and Cu(I) can be measured spectrophotometrically at 750 nm. A calibration curve can be obtained by dissolving known amounts of stratum corneum in 1 M sodium hydroxide. A piece of tape that has not been in contact with skin is subjected to an identical procedure and serves as negative control. The method was recently adapted to a 96-well plate format, notably reducing analysis times [129],... 

Annapurna et al. [13] established simple, accurate, and reproducible UV spectrophotometric methods for fhe assay of buclizine based on the formation of precipitation, charge transfer, and redox products. Precipitation/ charge transfer complex formation of fhe buclizine with I2/p-nitro methyl amino phenol sulfate-sulfanilic acid by method A, the precipitation/complex formation with ammonium molybdate/potassium thiocyanate by method B and precipitation/redox reaction of buclizine with phosphomolybdic acid/Co /EDTA by method C were proposed. Determination of buclizine in bulk form and in pharmaceutical formulations was also incorporated. 

A convenient method of assay of phosphorylase activity is based on the rate of liberation of inorganic phosphate from glucose-1-phosphate, i.e., phosphorylase is measured in the direction of glycogen synthesis [6], After a 10-min incubation at 37°C followed by precipitation with trichloroacetic acid, the supernatant fraction is analysed for inorganic phosphate by the method of Fiske and SubbaRow [163]. This involves the formation of phosphomolybdic acid from the phosphate present and the reduction of the phosphomolybdic acid to produce a blue colour whose intensity is proportional to the amount of phosphate present. 

Fig. 8 Malachite green assay for phosphate detection. The enzymatic reaction produces inorganic phosphate P. A secondary reaction is used to form a phosphomolybdate-malachite green complex, which has a strong absorbance at 660 nm.

Another common procedure for stopped assays is the use of molybdate under acid conditions to complex and spectrophotometrically assay inorganic phosphate. This provides a usefiil alternative to jMiitrophenyl phosphate for phosphatase assays, as physiologically relevant phosphate esters can be used. For example, this assay also provides a useful method for measuring ATPase activity. Details of the phosphomolybdate assay arc given here (however, see Section 8.5 for methodologies that utilize plate readers). 

A phosphomolybdate assay for inorganic phosphate released by ATPase activity... 

These results would confirm what was indicated in the previous item, i.e., that the P2Moj023 species dissociates as a consequence of adsorption and that, on the solid, the ratio of boA adsorbed P and Mo is different from that corresponding to the stoichiometry of such phosphomolybdate, while in the assays that used FK solutions, the adsorbed species is a phosphomolybdate. 

Total phospholipids are quantitated by determination of the phosphorus content of lipid extracts from which non-lipid phosphorus has been removed by purification procedures. For this purpose chloroform-methanol extracts subjected to diffusion purification (Folch et al. 1951, Sperry 1955) are suitable. Lipid phosphorus is determined in aliquots of these extracts after digestion. Most procedures for determination of phosphorus are based on the method of Fiske and Subbarow (1925) which utilizes conversion of phosphate to phosphomolybdate and its subsequent reduction to molybdic blue. Modifications of this method were reviewed by Lindberg andERNSTER (1956). Avery convenient phosphorus assay was described by Bartlett (1959). Total phospholipids are calculated by multiplication of the lipid phosphorus values with 25. These values are only approximations since phosphorus does not represent exactly 4% of each phospholipid molecule. 

The Lowry and BCA methods are related, in that the first step of the assay is the reduction of copper ion, Cu to Cu, by protein amides under alkaline conditions. In the Lowry method the reduced copper— and to a lesser extent some protein side chains— react with the Folin-Ciocalteu reagent (phosphomolybdate/pho-sphotungstate) to produce a blue color. In the BCA assay the BCA complexes with the Cu, which absorbs strongly at 562 nm. Although both the Lowry and BCA methods must be carefully timed and are subject to interference from buffer components, the BCA method is less susceptible to interference, especially by detergents. 

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