Re: Misc. - Alkylation

Steve Latshaw (latshaws@mis.finchcms.edu)
Tue, 27 Jan 1998 16:23:05 -0600

A long time ago, we had tried to modify methionines on purpose.
After blocking the Cys residues with DTNB the protein was reacted with
Iodoacetic acid for 30 minutes at a concentration of 37.5mM IAA and 60mM
protein at a pH 7. This gave us two methionines completely modified with the
protein in the native condition. The reactivity of methionines to IAA
doesn't change dramatically from pH 5-8 so you may be having some
modification there. We never had any methionine modification with vinyl
pyridine and its my understanding that Iodoacetamide is even more reactive
than Iodoacetic acid. You didn't mention what you used for reduction but
swamping the reaction with DTT prevents most side reactions but using
tributylphosphine doesn't.
Incidentally, we used 14C-IAA to follow our time course for
methionine reaction and we always bought the free acid and stored it without
neutralization as a 1M solution with cold IAA. The day that we did the
experiment we neutralized an aliquot of the stock with NaOH and discarded
any unused portion. I dug out one of the old vials today, its been in the
freezer for 15 years and its still not yellow.

Steve Latshaw
Chicago Medical School
latshaws@mis.finchcms.edu

At 03:39 PM 1/27/98 +1100, Ken Mitchelhill wrote:
>Colleagues,
>
>thank you to all those responding to my question about storage and
>purification of alkylating reagents, this message is a supplementary
>question on the same subject.
>
>We are doing ESI-MS experiments on whole recombinant proteins to establish
>disulphide bond formation. Our approach is to purify monomer away from
>oligomer by gel filtration chromatography and then perform either
>alkylation or reduction/alkylation and compare MW's by ESI-MS.
>
>We have the situation where a protein with three cysteines increases in MW
>by one alkylating group prior to reduction and by three alkylating groups
>after reduction, exactly what we were anticipating, one free cysteine and
>one disulphide.
>
>The problem is that there is another smaller (perhaps 5% by peak height)
>species in the spectra representing two alkylation sites prior to reduction
>and four after reduction.
>
>Does this mean that both the 4-vinyl-pyridine and the iodoacetamide are
>alkylating some other reside besides cysteine or is there some other
>explanation that has escaped me?
>
>Regards....Ken
>
>********************************
>
>Ken I. Mitchelhill
>The John Holt Protein Structure Laboratory
>St. Vincent's Institute of Medical Research
>41 Victoria Parade
>Fitzroy 3065 Victoria
>AUSTRALIA
>
>Telephone: 61-3-9288 2480
>Facsimile: 61-3-9416 2676
>
>Email: k.mitchelhill@medicine.unimelb.edu.au
>
>Laboratory: http://www.medstv.unimelb.edu.au/WWWDOCS/SVIMRdocs/JHPSL.html
>ABRF: http://www.medstv.unimelb.edu.au/abrf.html
>
>***********************************
>
>
>