I am working on Glycyl Glycine di peptidase (GGDP) enzyme activity assay, where GGDP hydrolysis glycyl glycine substrate releasing glycine as the product. I am using precipitation reagent (ethanol:water in 99:1 v/v) to stop the reaction followed by centrifugation and absorbance at 220nm recorded of the supernatant. How can I calculate the enzyme activity in umoles ggdp hydrolyzed/min/mg protein? Glycine 0.15M I am taking for standard curve.
Thank you in advance.
This assay I am doing using cell lysate, assuming the cells I am using contain this enzyme- ggdp.
I don't think the method you are planning to use is going to work. There will be far too much background absorbance in the cell lysate to detect a change in absorbance at 220 nm. This background will include Gly and other amino acids that were already present in the cells.
You need an assay that will specifically detect Glycine produced from Gly-Gly, even in the presence of excess Gly-Gly, preexisting Gly, and everything else in the cell lysate. This is a case where the best method would probably be to use radioactive Gly-Gly and to follow the reaction by the production of radioactive Gly. This could be done by thin-layer chromatography or HPLC with a radioactivity detector to separate Gly from Gly-Gly.
Thank you for your suggestion @Adam B Shapiro but I won't be able to proceed with radioactive gly.
You could try using a coupled enzyme detection system using L-amino acid oxidase.
https://www.sigmaaldrich.com/US/en/product/sigma/a9253
https://worthington-biochem.com/LAO/assay.html
220 nm is more specific for amide bond (peptide bond) so you can observe disappearing absorbance during your assay. If you want to do calibration curve you should use Gly-Gly dipeptide rather than Gly, which does not absorb at 220 nm significantly.
You will find data for 214 nm in:
Article Prediction of Molar Extinction Coefficients of Proteins and ...
According to this paper, molar extinction coefficient for the peptide bond is 923 and for Gly is 21.
Dear Dr Shikha Chaudhary . See the following useful link: https://core.ac.uk/download/pdf/76215687.pdf
Kindly see also the following very good RG link: Article Prediction of Molar Extinction Coefficients of Proteins and ...
See also the following useful link: https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0006-Extinction-coefficients.pdf
Check https://pubmed.ncbi.nlm.nih.gov/17539659/
https://www.sciencedirect.com/science/article/pii/S030881460300219X.
Also check https://www.jocpr.com/articles/molar-extinction-coefficients-of-some-amino-acids.pdf
https://link.springer.com/content/pdf/10.1023%2FA%3A1020453016132.pdf
Use the formula in this paper
https://www.biosyn.com/tew/How-is-Extinction-Coefficient-Determined-for-Proteins.aspx
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