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Pyrophosphate Fluorometric (Blue) Assay Kit, EnzChek® from Molecular Probes (Invitrogen)

ProductsPyrophosphate Fluorometric (Blue) Assay Kit, EnzChek® from Molecular Probes (Invitrogen)
Company Molecular Probes (Invitrogen)
Item Pyrophosphate Fluorometric (Blue) Assay Kit, EnzChek®
Description Molecular Probes EnzChek Pyrophosphate Detection Kit provides a fast, convenient and inexpensive spectrophotometric method for measuring the inorganic pyrophosphate (PPi ) produced by a number of biochemical reactions, such as DNA and RNA polymerizations, cyclic AMP formation by the enzyme adenylate cyclase and the enzymatic activation of fatty acids to form their coenzyme A esters.1 The EnzChek Pyrophosphate Assay Kit is a modification of our EnzChek Phosphate Assay Kit (E-6646), which is based on the method originally described by Webb.2 In the presence of inorganic phosphate (Pi ), the substrate 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) is converted enzymatically by purine nucleoside phosphorylase (PNP) to ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine (Figure 1). Enzymatic conversion of MESG results in a shift in absorbance maximum from 330 nm for the substrate to 360 nm for the product.

The EnzChek Pyrophosphate Assay Kit includes the enzyme inorganic pyrophosphatase, which catalyzes conversion of PPi present into two equivalents of Pi. The Pi is then consumed by the MESG/PNP reaction and detected by an increase in absorbance at 360 nm. Additional sensitivity is gained by the amplification of one molecule of PPi to two molecules of Pi. The assay can detect as little as 1 nanomole of PPi in 1 mL reactions (Figure 2) and can be performed over a pH range of 6.5 to 8.5.2 The MESG/PNP reaction has been used for continuous spectrophotometric determination of aminoacyl-tRNA synthetase activity.3 In addition, we have tested the ability of this assay to detect the PPi liberated by the activities of acetyl-CoA synthetase (Figure 3) and luciferase (Figure 4). In these experiments, we have detected as few as 5 10-5 U of acetyl-CoA synthetase activity and 10-2 U of luciferase activity.

Info Molecular Probes (Invitrogen)Molecular Probes (Invitrogen)
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