Inorganic pyrophosphatase catalyzes the hydrolysis of pyrophosphate into two molecules of phosphate. It is inactive organic pyrophosphates and organic phosphates. This enzyme is used to enhance molecular biology reactions where pyrophosphate production is limiting, such as high-yielding in-vitro transcription. This product contains wild-type pyrophosphatase A, encoded by E. coli's ppA gene. It is produced from a recombinant source.
- E. coli pyrophosphatase A
- Activity > 10 U / mg of lyophilizate
- Phosphatase undetectable at < 30 micro-Units per U of pyrophosphatase
- Protease undetectable at < 0.6 ng Proteinase K equivalents per U of pyrophosphatase
- DNAse undetectable at < 10 milli-Kunits-Units per U of pyrophosphatase
- RNAse < 1 milli-Kunits-Unit per U of pyrophosphatase
Storage & Handling
The lyophilizate is stable under many conditions, including ambient shipping. Upon arrival, it can be stored in the fridge or freezer. To use pyrophosphatase in a reaction, one can weigh in fresh powder each time, if balance accuracy permits.
The lyophilizate can be dissolved in 50% glycerol in water, to create a liquid buffer for long term storage. Many storage buffer recipes one can find on the internet or in publications will work fine. The pyrophosphatase works best in the presence of 1 mM fresh DTT. If the pyrophosphatase has been stored for a very long time, or the characteristic (mildly stinky) odor of the DTT appears to have disappeared, fresh DTT should be added.
One unit of pyrophosphatase is defined as the amount of enzyme that produces 1 umol of phosphate per minute from 50 uM pyrophosphate in 20 mM Tris pH 8.2 + 2 mM MgCl2.
We measure the amount of phosphate produced in the reaction using the Malachite Green method. It produces a complex with phosphate, but not pyrophosphate that absorbs at 620 nm. Here we compare our enzyme with that from a major US vendor, whom we will politely call "Vendor X". Both enzymes are used at 62.5 micro-Units in a 50 ul reaction, as per each Vendor's specification:
The phosphate standard curve can be used to calculate each enzyme's activity directly. The results of that were:
GACT units in the reaction: 174 micro-Units; Vendor X units in the reaction: 118 micro-Units. Thus, based on this experiment, both vendors provide more enzyme than they advertise. As measured on this individual day, GACT provided 2.79 fold more, and Vendor X provided 1.89 fold more than expected.
This extra provision is standard industry practice. The specific amounts are simply choices made by the vendors. It is probably not due to any fundamental difference between the enzymes themselves. If your application is sensitive to slight excess activity, then it will generally be useful to perform your own activity assay, regardless of where your enzyme comes from.