10.1021/bm400643p.s001
Jason Kerr
Jason
Kerr
Jessica
L. Schlosser
Jessica
L.
Schlosser
Donald R. Griffin
Donald R.
Griffin
Darice
Y. Wong
Darice
Y.
Wong
Andrea M. Kasko
Andrea M.
Kasko
Steric Effects in Peptide and Protein Exchange with
Activated Disulfides
American Chemical Society
2013
kDa
MW
pH
thiolate
disulfide size
protein
Activated DisulfidesDisulfide exchange
2013-08-12 00:00:00
Journal contribution
https://acs.figshare.com/articles/journal_contribution/Steric_Effects_in_Peptide_and_Protein_Exchange_with_Activated_Disulfides/2388544
Disulfide exchange is an important
bioconjugation tool, enabling
chemical modification of peptides and proteins containing free cysteines.
We previously reported the synthesis of a macromer bearing an activated
disulfide and its incorporation into hydrogels. Despite their ability
to diffuse freely into hydrogels, larger proteins were unable to undergo
in-gel disulfide exchange. In order to understand this phenomenon,
we synthesized four different activated disulfide-bearing model compounds
(<i>M</i><sub>n</sub> = 300 Da to 10 kDa) and quantified
their rate of disulfide exchange with a small peptide (glutathione),
a moderate-sized protein (β-lactoglobulin), and a large protein
(bovine serum albumin) in four different pH solutions (6.0, 7.0, 7.4,
and 8.0) to mimic biological systems. Rate constants of exchange depend
significantly on the size and accessibility of the thiolate. pH also
significantly affects the rate of reaction, with the faster reactions
occurring at higher pH. Surprisingly, little difference in exchange
rates is seen between macromolecular disulfides of varying size (<i>M</i><sub>n</sub> = 2 kDa – 10 kDa), although all undergo
exchange more slowly than their small molecule analogue (MW = 300
g/mol). The maximum exchange efficiencies (% disulfides exchanged
after 24 h) are not siginificantly affected by thiol size or pH, but
somewhat affected by disulfide size. Therefore, while all three factors
investigated (pH, disulfide size, and thiolate size) can influence
the exchange kinetics and extent of reaction, the size of the thiolate
and its accessibility plays the most significant role.