.&frcrs. Vol. 35. No. IO, pp. 1587-1588. 1994 EISwk !ScieauXLAd RintCdinorutBritlin lM404039/94 $6.oo+o.00
of Protein SuIfhydryl Helen
Department of Biokogical Sciences, University of Waikato. Hamilton, New Zealand
Abstractz The detection of sulfhydryl groups in proteins is limited by the inaccessibility of the sulfhydryls to the reagent, 5,5’ditbiobis(2-nitrokrobenzoic acid). The addition of otlwdisulfidcs such as 2,2’dithiobis(etbylamine) to the chromophoric disulfide increases both the rate and the extent of detection of “buried” sulfhydryl groups in pmtcins.
The relevance of sulfbydryl groups and disulfide bonds to the structure and function of proteins has led to the need for accurate means of quantifying dithiobis(2-nitrobenic
the sulfbydryl groups present in the protein.
acid) abbreviated as DTNB, reacts with sultbydryl
product, the 2-nitm-Qhiobenzoic
Ellman’s reagent, 5,5’-
gmups and releases a chromophoric
acid anion, which can be used to detect and quantify the number of sulfhydryl
groups present in a protein sample. However, this technique is limited to the detection of unhindered, solventaccessible sulflrydry~ groups. Thus, in a protein molecule. even after its denaturation as guanidinium
with chaotropic agents such
chloride or urea, many sulfltydryl functions remain inaccessible to reaction with DTNB and are
thus not detected’?*. The accuracy with which sulfhydryl groups can be detected, even in denatured proteins, needs to be improved.
The use of rapidly reacting, more permeable disulfide groups such as cystamine,
to carry out a primary reaction with protein sulfhydryl groups to release a readily-
for reaction with DTNB will be described.
Free sulfhydryl groups were determined by adding either DTNJ3 (0.5 mM) or the disulfide. cystamine (0.4 mh4 or 4.0 n&f) or a mixture of both reagents using the procedure as deskbeds. The number of sullbydryl tbe addition of urea or gdm-Cl.
groups measured by DTNB increases as the protein is more fully denatured by Although DTNB reacts quantitatively
with free cysteine-HCI,
number of sulfbydryl groups measured for each protein, even under denaturing conditions, is consistently the number predicted by the primary peptide sequence.
MNl3 is known to react only with sulfbydryl groups
accessible to the hydrophilic external environment *v4. Thus, a proportion of the sulfhydryl groups, even in denatured proteins, are still buried in stericahy-hindered
The chemical structure of DTNB containing both au aromatic ring and a negatively charged carboxylic acid may limit its reactivity in certain protein environments. A disulfide which is far more reactive than DTNB and is of sufficiently different chemical structure may react with the protein sulfbydryl and form the RS- anion in solution which can then go on to react with Dl’NB to form the colored assayable nitrobenzoate
anion. By this
series of reactions, the second disulfide does not need to produce a colored anion, but merely needs to be much more reactive than the DTNB with the previously inaccessible protein sulfhydryl.
is present in the muction vessel with DTNB, the number of sulfbydryl
be detected increaws by as much as 10X for ov~bumin between different protuins.
residues which can
the magnitude of the “cy~~~~~
This difference may be attributed to the different chemical environment
sulfhydryl residues in different proteins.
For example, ovalbumin
contains four -SH groups5 of which only 0.2
-SH is detected in the presence of DTNB while 2.0 -SH are detected in the presence of both DTNB and cystamine.
ht contrast, 8-l~toglo~lin
contains only one -SH grou#
which is detected equally well in the
presence of DTNB or both DTNB and cystamine. The utility of a second, more reactive disulfide binding to DTNB-inaccessible dependent on the slower rate of reaction of DTNB. Cystamine, fifken times more rapidly than DTNB with BSA sul~yd~ls
sulfhydryl groups is
a positively charged disulfide, is known to react
6, Protein sulf’hydryls which do not readily react
with DTPTB (either because of its negative charge or arom,aticity) may be mom reactive towards the positively charged cystamine.
studies will examine the “cystarruneeffect”
for sulfbydryls with known, chemical
At present, the addition of cyatamine to aid the reaction of DTNB in native protein structures is
recommended. Acknowledgementa. acknowledged.
support from the University
of Waikato Research Committee
I especially thank F. Cochrane for technical assistance.
References Femandez-Diex, M.J.; Osuga, D-T.; Feeney, R.E. Arc& Biock. Biophy. 1964,107,449-458. 1. Torchinsky, Y.M. [email protected]
in Proteins; Pergamon Press: Oxford. 1981. 2. Riddles, P.W.; Blakeley, R.L.; Zerner, B. Methods Enzymol. 1983,91,49-61. Ellman, G.L. Arch. 3. Birch. Binphys. 1959.82, 70-77. 4. Clayshulte, T.M.; Taylor, D.F.; Hcnzl, M.T. J. Biol. Chem. 1990,265, 1800-l 805. Barker, W.C. [email protected]
Sequence Data Base., Release 8. Is6 National Biomedical Research Foundation, I5 . G~rgetown University Medical Center, W~hingt~, DC. Wilson. J-M.; Wu, D.; Motiu-DeGrood, R.; Hupe, D.J. J. Am. Chem. 5’0~. 1980,102, 359-363. 6.
Weceived in UK 15 November 1993; revised 31 December 1993; accepted 7 &uuuy