I want to label viral capsids with a certain heavy metal
cluster. The capsid consists of 240 protein monomers with
one engineered cysteine per monomer. The thiols are
surface accessible. Capids are in 100 mM HEPES buffer at
pH 6.9.
The Hg reagent is Tetrakis(acetoxymercuri)methane. This
reagent has a central carbon atom surrounded by 4 Hg
atoms each with an acetate group. For use as a monovalent
reagent Tetrakis must first be reacted with three
equivalents of N,N-dimethyl-2-aminoethanethiol. Clearly
this gives a distribution of species with zero to four
thiolate groups.
When the reagent prepared above is added to a solution of
capsids there is rapid formation of a ppt, even when used
in submolar equivalents. Reagent added to HEPES buffer
alone does not form a precipitate suggesting that it is
not a buffer problem but rather a cross-linking of
capsids by multivalent reagent species. However, equal
concentrations of capsids without the cysteine are also
precipitated by the reagent, and in fact even more so
suggesting that cross-linking is not via thiols. At pH
6.9 the reactivity of amino groups should be very low.
Can this reagent react elsewhere?
Any suggestions as to why the precipitate forms and how
to avoid it? Thanks for any advice or leads.
Norman Watts