I am planning to study enzyme kinetic on acid phosphatase activity on isolated phosphate solubilizing bacteria which have been isolated from paddy field soil with pH ranging between 5-6.5. Since i am only a beginner researcher can anyone give me some tips and guidelines for this.
8/11/16
Dear Lalrampani,
Acid phosphatases (AP) are relatively easy to assay. A convenient substrate is para-nitro-phenyl-phosphate (PNPP) which is available from Sigma and other chemical suppliers. This is a man-made, artificial substrate that AP breaks down to release inorganic phosphate (Pi) and para-nitro-phenol (PNP). After a suitable incubation period and at a suitable temperature and pH, the reaction is stopped by addition of NaOH. This inactivates the enzyme and raises the pH to ~12-13. The high pH is necessary because PNP is essentially colorless at pH
The method cited by Bill Colonna is a good one; the Sigma method follows that in the 2nd edition of Bergmeyer which my lab has used with success in the past.
The one downside is that it is a so-called "single point" reaction and you are unable to follow the catalytic rate over time, which may be inconvenient if you wish to study the kinetics of your particular acid phosphatase.
There are a couple of other substrates and conditions that allow continuous monitoring of the reaction rate over time; see the paper by Klaus Lorentz:
http://www.sciencedirect.com/science/article/pii/S0009898102002954
or that from Renze Bais:
http://www.clinchem.org/content/22/12/2025.full.pdf+html
Alpha-naphthyl phosphate can be coupled with Fast Red dye and has been used on automated analyzers for measurements using human serum:
http://www.sciencedirect.com/science/article/pii/0009898177900250
I'm not sure what exactly what you want to do, but if you're studying reaction kinetics, continuous monitoring reactions are usually the way to go.
Bob Rej
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology