Both pH and time of exposure are critical in alkylation. Residues
other than Cys that can be alkylkated are His, Lys, and Met. It is the
nucleophilic form that is alkylated, that means the unprotonated form and
thus the importance of pH (Met is pH independent). Cys is the most reactive
while the others are usually much slower unless they are in an environment
that activates them. So the take home here is do it fast at the right pH.
My suggestion is to dissolve in 6 M guanidine hydrochloride, adjust the pH
to 8.0, add 4-VP (make sure it is very fresh) incubate for only 30 seconds
to a minute. At this point you need to get the 4-VP out of there before the
slower reactions become substantial, either by rapid solvent exchange of
some kind (ultracentrifugation?) or by killing the 4-VP with beta
mercaptoethanol before dialysis. The latter however, will probably reduce
the disulfides. Which is OK if you can be sure that this will happen slower
than the consumption of 4-VP. Since BME is not a great reducing agent
(compared to DTT), this may be the case but there are no guarantees. A
potential approach is to do the same thing with a completely reduced sample
and then compare the results for differences.
Another place to look is chapter 15 in Current Protocols in Protein
Science.
Hope this helps.
Gregory A. Grant
ggrant@pharmdec.wustl.edu