Inorganic pyrophosphatase assay

Vance DH, Czamik AW (1994) Real-time assay of inorganic pyrophosphatase using a high-affinity chelation-enhanced fluorescence chemosensor. J Am Chem Soc 116 9397-9398... 

Lieberherr, M., Vreven, J., Vaes, G. The acid and alkaline phosphatases, inorganic pyrophosphatases and phosphoprotein phosphatase of bone. I. Characterization and assay. Bio-chim. Biophys. Acta 293, 160 (1973)... 

Recently, Czamik et al. have reported the use of the acyclic protonated amine host 9 as a chemosensor of pyrophosphate. Typical fluorescence sensing methods rely on the ability of a complexed anion to quench the fluorophore. The fluorescence intensity of host 9, however, is actually increased upon complexation of anions and its 2200-fold selectivity of pyrophosphate over phosphate allows for real-time assay of pyrophosphate hydrolysis by inorganic pyrophosphatase.18... 

Vance and Czamik have also developed a new chemosensor for pyrophosphate based on an anthracene chromophore (Figure 91) [118]. This compound exhibits low fluorescence. However, upon binding pyrophosphate, the fluorescence increases considerably. It has been shown by Vance and Czamik that inorganic pyrophosphatase can be assayed in real time using this chemosensor since the inorganic phosphate produced does not affect the fluorescence of this molecule. 

U/ml inorganic pyrophosphatase from Baker s yeast (Sigma-Aldrich, St. Louis, MO, USA). Resuspend 500 U lyophilized enzyme and buffer salts in 250 pi water. Store at -80 °C in 5 pi aliquots. Store thawed aliquot on ice and discard at the end of assay day. 

Dick and Tabatabai " have described an assay for inorganic pyrophosphatase activity in soils based on a specific colorimetric determination of extracted orthophosphate after its release from inorganic pyrophosphate (0.05M) incubated with soils for 5h at 37°C. Soil suspensions were buffered at pH 8.0. In the absence of toluene, the amounts of orthophosphate released g soil were proportional to incubation time (up to 7h), and for a given time to the amounts of soil added. Activities were unaffected by toluene addition to soils and toluene was omitted from the recommended assay procedure. 

Scheme II. Reaction scheme rationalizing the observed fluorescence assay of inorganic pyrophosphatase activity using chemosensor 3.

A flow assay was reported for determination of inorganic pyrophosphate a pyrophosphatase was coimmobilized with luciferase on Sepharose beads with continuous flow of saturating concentrations of substrates. The instrument allowed automation with a throughput of approximately one sample every 4 min. 

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