Thermostable Pyrophosphatase

• Hydrolysis of inorganic pyrophosphate to produce orthophosphate
• Low K_m (5.4 µM)
• Active at pH 7–9
• Optimal temperature for activity at 75°C

Thermostable pyrophosphatase is a recombinant enzyme from Sulfolobus acidocaldarius, which catalyzes the Mg²⁺-dependent reaction of P₂O₇^-4 + H₂O → 2HPO₄^-2.  It has a low K_m (5.4 µM) for pyrophosphate, is active between pH 7 and 9, has an optimal temperature for activity at 75°C and is functional under PCR conditions (1-3). 

Supplied in:
10 mM Tris-HCl, 50 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol (pH 7.5 at 25°C)

• Storage temperature: –25°C to –15°C
• Molecular weight: 19.4 kDa

The protein is produced by an E. coli strain carrying the Thermostable Pyrophosphatase gene from S. acidocaldarius.

One unit is the amount of enzyme that will liberate 1 µmol of phosphate per minute from inorganic pyrophosphate at 75°C and pH 8.5.

Usage Instructions 

The Thermostable Pyrophosphatase can reduce the inhibitory effects of accumulated PPi in polymerase chain reactions (PCRs) (5-7). As a starting point for PCR, add 0.01-0.1 U/µL of Thermostable Pyrophosphatase to identify the optimal concentration. 
 
Notes:  
Thermostable Pyrophosphatase catalysis is Mg^2+-dependent. Therefore, it is important to have Mg²⁺ in the reaction buffer. It is compatible with most PCR buffers that contain Mg2⁺. 

Quality Control 

Unit activity

The assay is based on that described by Taussky and Shorr (4). Briefly, 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 75°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 molybdate/ferrous sulfate reaction. 
 
Protein concentration (OD₂₈₀) 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 protein of interest band 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 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 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 16S rDNA contamination is evaluated using 5 µL replicate samples of enzyme solution 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 product is available for molecular biology applications such as:

• Production of orthophosphate
• Avoiding inhibition in RNA synthesis and modification reactions
• Avoiding inhibition in PCR reactions

References

1. Leppänen, V.M. et al. (1999) Protein Sci., 8(6):1218-31. 
2. Hansen, T. et al. (1999) Arch. Biochem. Biophys., 363(1):135-47.
3. Meyer, W. et al. (1995) Arch. Biochem. Biophys., 319(1):149-56.
4. Taussky, H.H. and Shorr, E. (1953) J. Biol. Chem., Jun;202(2):675-85. 
5. Moldes, C. et al. (2004) Appl. Environ. Microbiol., 70(8): 4642-4647. 
6. Dignam, J.D. and Deutscher, M.P. (1979) Biochemistry, 18(14):3165-70. 
7. Gibson, N.J. et al. (1997) Anal. Biochem., 254(1):18-22.