You want to map the H2A acetylation site for your HAT a reco

You want to map the H2A acetylation site for your HAT (a recombinant histone acetyltransferase, which transfers an acetyl group from acetyl-CoA to a Lys side chains on histone H2A), so you need to make a proteolytic digest of the modified H2A for HPLC-MS analysis. You start with 10 mg of modified H2A.
You buy chymotrypsin from Sigma, 5 mg with a specific activity of ~50 U/mg. How much chymotrypsin do you need and how long will it take?

i) Estimate a value for the conversion mg of H2A to mol of substrate. From last semester, the average MW per amino acid residue (weighted by frequency of different amino acids in proteins) is 110 daltons. H2A contains 129 residues.

ii) There are approximately 20 chymotrypsin sites in the polypeptide sequence of H2A. How many mol of H2A substrate were you were given?

iii) For the digest to be complete in an hour, how much chymotrypsin (in g) do you need to add?

Solution

- we only need 1mole of chymotrypsin because for 40-60U/mg specific activity, 1 unit hydrolyzes 1mole per min at ph 7.8 at 25 degree celcius.

(i) 9 amino acids would be around 990 Da, which you could round up to 1 kDa. ONE amino acid is 110 Da. Once you know that, you can easily figure out the by multiplying the number of amino acids in it by 110 Da!

similarly, 110*129=14190 daltons

(ii) 0.2mol were given

(iii) around 5g to make up the 1 mole and 1 mole of chymotrypsin can complete the experiment in an hour .