I'm degrading soy protein with a alcalase and I have been measuring base consumption (while regulating the pH at the enzyme pH optimum), which according to literature is proportional to the amount of peptide bonds that are calved. So I have these curves (base consumption - time, calculated %DH - time), and I can get "initial reaction velocity" from these curves if I add a linear fit with the first few points.
I would like to calculate the time I need to regulate the pH, and then let it drop, so I would get a desired pH value at a desired time point.
I did a few experiments so I have an idea how the pH drops, but I would like to have a calculation, because this should be easy.
any idea how to elegantly solve this?
Recall that pH = -log [H+]. If the solution in which your reaction occurs is unbuffered, then you can, in principle, calculate what the pH will be at any future time based on the known rate of H+ production. In reality, non-ideal circumstances may prevent this from working, examples being (1) the solution is not really unbuffered because the protein itself and its hydrolytic products can act as a buffer due to the presence of ionizable side chains, (2) the enzyme activity changes as the pH changes, and (3) the reaction slows down as the substrate is consumed.
There may be a calculation that fits your kinetic model but subtilisin cleavage could be a difficult reaction to control in order to have consistent results. Using subtilisin for characterizing other proteins by digestion can yield somewhat variable results. This enzyme's degree of hydrolysis and specificity seems to vary significantly with pH, temperature, substrate concentration, enzyme concentration, end-product inhibition, (self-digestion?), mixing, etc. Can all the appropriate conditions be held constant as the titration is done? The pKa of any buffering solutes will vary with temperature and therefore the pH will vary. Is the addition of base performed in a non-denaturing manner and where dilution effects are not significant? Is it possible to measure an orthogonal parameter reflective of the kinetics other than pH such as a structural feature that is pH dependent in the presence of substrate?
Thank you all. I really appreciate your answers. Yes you are right, this is a really complex system. Of course the pH, enzyme activity, substrate concentration etc..., change during the reaction. We'll need to figure it out experimentally.
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