Fluorescence pyrophosphatase

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... 

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... 

Figure 25 A plot of f and f for selenium as a function of energy, (a) A theoretical plot from 5 to 20 keV. Reproduced from Ethan Merritt s website, with permission from Ethan Merritt, (b) A fluorescence scan of the selenomethionyl derivative of the CBS domain of Clostridium perfringens inorganic pyrophosphatase (H. Tuominen, unpublished) around the absorption edge showing energies where peak (red) and inflection point (blue) data were collected. The values of f" and f were fitted by the program CHOOCH.27...

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. 

PET sensor for dihydrogen pyrophosphate dianion. Both of these cases require careful pH control for success. Also, sensor B may be used as an assay for the pyrophosphatase enzyme since the hydrolysis products will be released from the receptor pair with concomitant loss of fluorescence. 

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

Figure 2. Fluorescence intensity as a function of time during the pyrophosphatase-catalyzed hydrolysis of PPi to 2Pi, as monitored using chemosensor 3.

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