32 M. H. Akabas
(Akabas et al. 1992 ; Karlin and Akabas 1998 ). One can factor out the influence
of the electrostatic interaction between the reagents and the cysteine thiolate by
comparing the relative rates to those observed with 2-ME (Stauffer and Karlin
1994 ).
An important caveat regarding the use of the MTS reagents is that they hydro-
lyze in water due to attack by hydroxyl ion. The rate of hydrolysis is dependent on
both pH and the charge of the MTS reagent (due to the electrostatic interactions
with OH− similar to those described above for thiolates). At pH 7.0 the hydrolysis
t1/2 ranges from ~ 10 min for MTSET to ~ 370 min for MTSES (Karlin and Akabas
1998 ). This raises an important issue of how to prepare stock solutions of MTS re-
agents. In unbuffered solution, reagent hydrolysis causes a rapid decrease in the pH.
This acidification effectively slows the hydrolysis to negligible rates on a time scale
of a day. So, many investigators will dissolve in water an amount of MTS reagent
for a day of experiments and store it on ice. Immediately prior to each experiment,
the aqueous stock solution can be diluted into the appropriate buffer and used in a
time frame compatible with the hydrolysis rate.
Another important issue to consider with the MTS reagents is that the covalent
disulfide bond they form with cysteine thiols is readily reduced by commonly used
reducing agents such as 2-ME, DTT, or TCEP. It can also be reduced by endogenous
cytoplasmic thiols such as glutathione or cysteine.
Six Protein Data Bank structures show MTS modified cysteines, one with multi-
ple MTSET-modified Cys residues (PDB # 3TBE, Streptococcus agalactiae sortase
C1) and five with MMTS (3PF3, 3H6S, 3KSE, 3KKU, 3KFQ). One of the MTSET-
modified S. agalactiae sortase C1 Cys is illustrated in Fig. 2. Note that the Cys
residue sulfur is on the water accessible surface in the surface dot representation and
the S-ethyltrimethylammonium extends above the protein surface (Fig. 2a).
2.2 p-Chloromercuribenzenesulfonate and Other Mercurials.
pCMBS was originally described as a negatively charged, membrane impermeant
mercurial compound (Vansteveninck et al. 1965 ) (Fig. 1b). An advantage of pC-
MBS over the MTS reagents is that pCMBS does not hydrolyze. However, pCMBS
does not discriminate as effectively between thiolate and thiol. Its reaction rate
with the thiolate is only 3400 times faster than with the thiol (Parikh et al. 2011 ).
In solution, pCMBS reacts orders of magnitude faster with simple thiols than the
MTS reagents. The second order reaction rate constant for pCMBS with cysteine is
1.2 × 109 M−1^ s−1 (Parikh et al. 2011 ), similar to rates measured for p-mercuribenzo-
ate (pCMB) (Hasinoff et al. 1971 ); Whereas the second order reaction rate constant
for MTSES with 2-mercaptoethanol is 1.7 × 104 M−1 s−1 (Karlin and Akabas 1998 ).
pCMBS has been used to modify endogenous cysteine residues in x-ray crystal-
lography to create heavy metal derivatives. These crystal structures show that the
–S-Hg-C- bond axis is essentially linear (PDB #: 1XZC, 1BH9, 1HDK, 1HJ1, and
1YP2) (Fig. 2b).