I am working on designing a reversible inhibitor for salivary aldehyde dehydrogenase and came across lots of chemical compounds that would probably be inhibitors of this enzyme, so these inhibitors might be able to modify this enzyme.
I want to know what the experiments are for cysteine modification.
Please give me a good suggestion - it will be highly appreciated.
Thanks.
If you want to see if the cysteine residues are modified, mass spectrometry experiment after tryptic digestion of the enzyme can be very useful (you can even identified the Cys residue). If you do not have access to mass spectrometers, you could measure cysteine residues before treatment using DTNB and after treatment and see if any difference in the number of cysteine residues available. You will in that case know if cys residues are modified but you will not be able to identify which ones.
If you don't have enough protein to use DTNB to measure the amount of free thiol in a protein sample, you can use Thio-Glo reagent, which is more sensitive. Obviously, this technique can not be used in the presence of free thiols like dithiothreitol or mercaptoethanol.
If you just want to know whether a compound forms a covalent bond with the protein, measure its mass by MS with and without treatment with the compound. If you want to test whether the reaction is with Cys, block the Cys residues first with N-ethylmaleimide or other thiol-reactive chemical.
If you want to see whether a compound blocks the reactivity of a particular Cys residue, you can react the protein, with and without the compound present, with a fluorescent maleimide or alkyl halide reagent, then digest the protein with trypsin, and run the peptides on an HPLC peptide mapping column to separate the peptides and quantify the fluorescence of each one using a fluorescence detector. Combining this with mass spectrometry allows the peptide peaks to be identified.
You're asking for experiments to modify Cys or to determine which Cys are modified?
EDIT - OK, now I've read again the title and I see it. Sorry
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