Reactivity of Protein sulfhydryl groups with disulfides

Reactivity of Protein sulfhydryl groups with disulfides

T&&-&M .&frcrs. Vol. 35. No. IO, pp. 1587-1588. 1994 EISwk !ScieauXLAd RintCdinorutBritlin lM404039/94 $6.oo+o.00 oo40-#39(94)EOO85-C Reaetivity o...

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.&frcrs. Vol. 35. No. IO, pp. 1587-1588. 1994 EISwk !ScieauXLAd RintCdinorutBritlin lM404039/94 $6.oo+o.00



of Protein SuIfhydryl Helen


with Disulfides

H. Petach*

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-

accessible solution-sulfhydryl

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,

the maximum

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.



When cystamine

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

of the

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.

Continuing environments.

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

is gratefuIly

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