E. coli Pyrophosphatase

• Catalyzes the hydrolysis of inorganic pyrophosphate to form orthophophate

E. coli Pyrophosphatase catalyzes the Mg-dependent reaction of P₂O₇^-4 + H₂O → 2HPO₄^-2.

This enzme is supplied in 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA and 50% glycerol.

SDS available upon request.

Enzyme properties

• Storage temperature: –25°C to –15°C
• Molecular weight: 19,704 Daltons

Source of recombinant enzyme protein
The protein is produced by a recombinant E. coli strain carrying the E. coli Pyrophosphatase gene.

Unit definition: E. coli Pyrophosphatase catalyzes the Mg-dependent reaction of P₂O₇^-4 + H₂O → 2HPO₄^-2.

Source of protein
The protein is produced by a recombinant E. coli strain carrying the E. coli Pyrophosphatase gene.

Unit definition
One unit is the amount of enzyme that will liberate 1 µmol of phosphate per minute from inorganic pyrophosphate at 37°C and pH 8.5

References

1. Lahti, R., Pitkäranta, T., Valve, E., Ilta, I., Kukko-Kalske, E., and Heinonen, J. (1988) Cloning and characterization of the gene encoding inorganic pyrophosphatase of Escherichia coli K-12. J. Bacteriol. 170:5901.
2. Baykov, A.A., et al (1996) Catalysis by Escherichia coli inorganic pyrophosphatase: pH and Mg2+ dependence. Biochemistry 16:4655.
3. Taussky, H.H., and Shorr, E. (1953) A microcolorimetric method for the determination of inorganic phosphorus. J. Biol. Chem. 202:675.

Quality control analysis

Enzyme dilutions are added to 30 mM Tris HCl (pH 8.5), 1.5 mM MgCl2 and 1.5 mM sodium pyrophosphate. After a 10 minute incubation at 37° C, the product formed, 2- orthophosphate, is reacted with ammonium molybdate to form phosphomolybdic acid. The phosphomolybdic acid is then reduced by ferrous sulfate under weak acidic conditions to form a blue color, the absorbance of which is measured at 660 nm. The amount of product formed is extrapolated from a phosphate standard curve generated from the ammonium molydate/ferrous sulfate reaction. The assay is based on that described by Taussky and Shorr (3).

Protein concentration is determined by OD₂₈₀ absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the band corresponding to the protein of interest in the diluted sample.

Single-stranded exonuclease is determined in a 50 µl reaction containing a radiolabeled single-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded exonuclease activity is determined in a 50 µl reaction containing a radiolabeled double-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded endonuclease activity is determined in a 50 µl reaction containing 0.5 µg of plasmid DNA and 10 µl of enzyme solution incubated for 4 hours at 37°C.

E. coli contamination is evaluated using 5 µl replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

• RNA modification