Sensitive quantitative analysis of disulfide bonds in polypeptides and proteins
- PMID:6547275
- DOI: 10.1016/0003-2697(84)90786-3
Sensitive quantitative analysis of disulfide bonds in polypeptides and proteins
Abstract
A sensitive quantitative method has been developed to determine the number of disulfide bonds in peptides and proteins. The disulfide bonds of several peptides and proteins were cleaved quantitatively by excess sodium sulfite at pH 9.5 and room temperature. Guanidine thiocyanate (2 M) was added to the protein solutions in order to denature them and thereby make the disulfide bonds accessible. The reaction with sulfite leads to a thiosulfonate and a free sulfhydryl group; the concentration of the latter was determined by reaction with disodium 2-nitro-5-thiosulfobenzoate (NTSB) in the presence of excess sodium sulfite. The synthesis, purification, and characterization of NTSB are described. The assay is rapid, requiring 3-5 min for oligopeptides and 20 min for proteins, and is as sensitive and quantitative as the sulfhydryl group assay employing 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent). It can be used for the analysis of as little as 10(8) mol of disulfide bonds, with an error of +/- 3%.
Similar articles
- Estimation of disulfide bonds using 2-nitro-5-thiosulfobenzoic acid: limitations.Damodaran S.Damodaran S.Anal Biochem. 1985 Feb 15;145(1):200-4. doi: 10.1016/0003-2697(85)90348-3.Anal Biochem. 1985.PMID:3839113
- A method for the controlled cleavage of disulfide bonds in proteins in the absence of denaturants.Kumar N, Kella D, Kinsella JE.Kumar N, et al.J Biochem Biophys Methods. 1985 Oct;11(4-5):251-63. doi: 10.1016/0165-022x(85)90007-7.J Biochem Biophys Methods. 1985.PMID:3840816
- Measurement of biological disulfides by postcolumn sulfitolysis following separation by HPLC.Crawhall JC, Kalant D.Crawhall JC, et al.Anal Biochem. 1988 Aug 1;172(2):479-83. doi: 10.1016/0003-2697(88)90470-8.Anal Biochem. 1988.PMID:3189788
- Disulfide bonds are necessary for structure and activity in Aspergillus ficuum phytase.Ullah AH, Mullaney EJ.Ullah AH, et al.Biochem Biophys Res Commun. 1996 Oct 14;227(2):311-7. doi: 10.1006/bbrc.1996.1506.Biochem Biophys Res Commun. 1996.PMID:8878514
- Measurement of mixed disulfides including glutathionylated proteins.Priora R, Coppo L, Salzano S, Di Simplicio P, Ghezzi P.Priora R, et al.Methods Enzymol. 2010;473:149-59. doi: 10.1016/S0076-6879(10)73007-X.Methods Enzymol. 2010.PMID:20513476Review.
Cited by
- Characterization of cyclic peptides containing disulfide bonds.Johnson M, Liu M, Struble E, Hettiarachchi K.Johnson M, et al.J Pharm Biomed Anal. 2015 May 10;109:112-20. doi: 10.1016/j.jpba.2015.01.009. Epub 2015 Jan 22.J Pharm Biomed Anal. 2015.PMID:25778927Free PMC article.
- Local interactions favor the native 8-residue disulfide loop in the oxidation of a fragment corresponding to the sequence Ser-50-Met-79 derived from bovine pancreatic ribonuclease A.Milburn PJ, Scheraga HA.Milburn PJ, et al.J Protein Chem. 1988 Aug;7(4):377-98. doi: 10.1007/BF01024887.J Protein Chem. 1988.PMID:3255371
- Structural disulfide bonds in the Bacillus thuringiensis subsp. israelensis protein crystal.Couche GA, Pfannenstiel MA, Nickerson KW.Couche GA, et al.J Bacteriol. 1987 Jul;169(7):3281-8. doi: 10.1128/jb.169.7.3281-3288.1987.J Bacteriol. 1987.PMID:3597322Free PMC article.
- Bacterial expression and characterization of human secretory class V phospholipase A2.Han SK, Yoon ET, Cho W.Han SK, et al.Biochem J. 1998 Apr 15;331 ( Pt 2)(Pt 2):353-7. doi: 10.1042/bj3310353.Biochem J. 1998.PMID:9531469Free PMC article.
- Spectroscopic, immunochemical, and thermodynamic properties of carboxymethyl(Cys6, Cys127)-hen egg white lysozyme.Denton ME, Scheraga HA.Denton ME, et al.J Protein Chem. 1991 Apr;10(2):213-32. doi: 10.1007/BF01024786.J Protein Chem. 1991.PMID:1930635
Publication types
MeSH terms
Substances
Related information
Grants and funding
LinkOut - more resources
Full Text Sources
Other Literature Sources