ORGLIST: Thio-Mercapto Reaction

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From: Norman Watts (Norman_Watts$##$
Date: Wed Oct 19 2005 - 12:50:13 EDT

Dear All,

I'm new to this List. My question is not strictly related to organic
chemistry, but rather to a thio-mercapto reaction. Nevertheless, I
suspect experienced advice is available from this group. Please bear
with me.

I wish to label a viral capsid protein with a phenylmercury derivative
(Molecular Probes Cat. No. H30462. Hg-Link Alexa Fluor 488
phenylmercury ). The protein forms obligate dimers, and 120 dimers
assemble to form a capsid with icosahedral symmetry. Each protein
monomer bears one cysteine, and on the capsid surface the cysteine thio
groups are exposed to the solvent and not in a position to form
disulfide bonds. We know this from the crystal structure.

The protein is prepared reduced with 5 mM dithiothreitol, and I remove
the reductant by gel filtration immediately prior to reaction with the
mercury compound. The solvent is 100 mM sodium phosphate, pH 7.4.

My uncertainties begin at this point:
Are the cysteine thiols likely to be in the form of mixed disulfides
after removal of the DTT?
Is there any benefit to further reducing the protein with immobilized
TCEP (tris(2-carboxyethyl)phosphine) after removing the DTT?
Is it likely that I don't have to worry about all this because the
mercury atom will simply displace anything on the sulfur atom anyway?

I could in principle explore all this but both protein and compound are
limiting, and I require a high occupancy of Hg-label on the capsid.

Thanks in advance,


Norman Watts, Ph. D.
National Institutes of Health
50 South Drive, Rm. 1509
Bethesda, MD 20892-8025
Phone: (301) 402-3418
Fax: (301) 480-7629

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