Question

You are developing an assay for a deaminase that catalyzes the following reaction, R-NH2 + H2O ® R-OH + NH3 To generate a measurable amount of product you include 0.5 µmol of the amine in a 100 µl reaction buffered by 25 mM HEPES-HCl, pH 7.25. For the primary amine, pKa = 7.25; for HEPES, pKa1 = 3, pKa2 = 7.55; for ammonium, pKa = 9.25. a. What fraction of the HEPES will be HEPESo (i.e., the protonated N form; see G&G Fig. 2-17) at the beginning of the reaction? b. If the concentration of the HEPES buffer is doubled, what fraction of the HEPES N will be protonated? Justify your answer. c. If all the amine is deaminated, what will be the final pH of the solution?

Answer 1

In this experiment from protein ammonia group is leaving and buffer is maintaining the pH range. As HEPES is a very good buffering reagent because it is a zwitter ion.

In assay deamination of reactant occurs and then there is a reaction with buffer solution.In starting the reaction rate is more as the site available are more. When there is change in temperature then the effect of HEPES become less.

So in this case, in the begining of reaction around 10% of HEPES get protonated.