Disulfide Bonds In Peptides Research Articles

Analysis for disulfide bonds in peptides and proteins

Publisher Summary This chapter discusses the analysis for disulfide bonds in peptides and proteins. Disulfide bonds make major contributions to the stability of the native conformations of proteins. The determination of the number of disulfide bonds per molecule is therefore crucial to structural studies of proteins. Many methods for the determination of disulfide bond concentration are proposed. Recently, a new method involving the use the reagent 2-nitro-5-thiosulfobenzoate (NTSB) is introduced. This method has significant advantages, the two most important being that it is both sensitive and quantitative. The reaction of NTSB with thiols and disulfides can be carried out in the presence of both dissolved oxygen and the reducing agent, sodium sulfite, which eliminates the inaccuracy and inconvenience associated with the necessity to work under an oxygen-free atmosphere as well as the need to remove the reducing agent. The NTSB assay is actually composed of two sequential reactions. The first reaction is the cleavage of a disulfide bond with sodium sulfite. The second reaction involves the nucleophilic attack of the thiolate produced in first reaction on NTSB to yield 1 mol each of a thiosulfonate and 2-nitro-5-thiobenzoate (NTB).

A new approach for detection and assignment of disulfide bonds in peptides

A new procedure is described for identification of disulfide bonds in peptides by fast atom bombardment mass spectrometry (FABMS). Prolonged bombardment of a disulfide-containing peptide in solution by a high-energy xenon beam results in gradual reduction of the disulfide bond. The reduction is the result of reaction intermediates initially produced by the xenon beam. The method for characterization of interchain disulfide bonds is based on the increase in the relative intensity of the pseudomolecular ions of the reduced peptides with a simultaneous decrease in the relative intensity of the protonated molecular ion of the oxidized peptide. This information allows one to identify peptide fragments covalently linked via intermolecular disulfide bonds. The intrachain disulfide bonds are identified by the increase in the relative intensity of the protonated molecular ion of the reduced peptide, relative to the intensity of the protonated molecular ion of the oxidized peptide. These results indicate that this method can be used to detect disulfide bonds of peptides and provides unambiguous information regarding disulfide bond assignment in peptides. Approximately 1 nmol of sample is required.

Estimation of disulfide bonds using 2-nitro-5-thiosulfobenzoic acid: Limitations

Recently an elegant method for the quantification of the number of disulfide bonds in proteins and peptides has been reported [ T. W. Thannhauser, Y. Konishi, and H. A. Scheraga (1984) Anal. Biochem. 138, 181–188]. The method is based on the quantification of 2-nitro-5-thiobenzoate (NTB) formed from the reaction of 2-nitro-5-thiosulfobenzoate with disulfides in the presence of excess sodium sulfite. Here it is reported that the NTB anion undergoes photochemical reaction with excess sulfite in the system, which results in the rapid disappearance of absorbance at 412 nm in the presence of light. The nonchromophoric derivative of this photochemical reaction is tentatively identified as a sulfo derivative of NTB. Based on these observations it is suggested that, for the quantification of disulfide bonds using the NTSB method, the assay should be carried out in the dark.

Sensitive quantitative analysis of disulfide bonds in polypeptides and proteins

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-thiosulfoben-zoate (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%.

ChemInform Abstract: SELECTIVE FORMATON OF THE DISULFIDE BOND IN PEPTIDE SYNTHESIS

Chemischer InformationsdienstVolume 12, Issue 49 Reviews ChemInform Abstract: SELECTIVE FORMATON OF THE DISULFIDE BOND IN PEPTIDE SYNTHESIS W. KOENIG, W. KOENIGSearch for more papers by this authorR. GEIGER, R. GEIGERSearch for more papers by this author W. KOENIG, W. KOENIGSearch for more papers by this authorR. GEIGER, R. GEIGERSearch for more papers by this author First published: December 8, 1981 https://doi.org/10.1002/chin.198149375AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume12, Issue49December 8, 1981 RelatedInformation

Direct Spectrophotometric Measurement of the Rate of Reduction of Disulfide Bonds: THE REACTIVITY OF THE DISULFIDE BONDS OF BOVINE α-LACTALBUMIN

Abstract The rate of reduction of the disulfide bonds of peptides and proteins by dithiothreitol can be determined by measuring, as a function of time, the absorbance at 310 nm of the oxidized dithiothreitol formed. The method is rapid and can produce a complete kinetic curve with 0.1 µmole of disulfide. Results obtained with oxidized glutathione, ribonuclease, bovine serum albumin and α-lactalbumin show that the correct stoichiometry is obtained for the reaction. All four of the disulfide bonds of α-lactalbumin are shown to be readily available for reduction by 10-3 m dithiothreitol in aqueous buffers at room temperature. At temperatures near 0°, three of the four disulfides are no longer susceptible to reduction. Thus the conformation of the α-lactalbumin molecule is altered in a dramatic way over this temperature range.

Investigation of Chain Reactions and Oxygen Effects during Radiolysis of Peptide Disulfide Bonds Using Cysteine-Glutathione Disulfide as a Model

γ-Radiolysis of cysteine-glutathione disulfide, the mixed disulfide of cysteine and the tripeptide glutathione, has been examined in unbuffered aqueous solutions (0.3 mM). In the presence of air the principal products are sulfinic and sulfonic acids of cysteine and glutathione and the symmetrical disulfides, cystine and glutathione disulfide. In the absence of air, cysteine, glutathione, and the sulfinic acid derivatives of these thiols were produced in moderate yields whereas the symmetrical disulfides were both formed in very high yields (G ∼10). Irradiation of solutions containing various concentrations of mixed disulfide and a range of oxygen concentrations showed that the high yields of symmetrical disulfides were due to a chain reaction which could be suppressed by oxygen. The following reactions were proposed: RS· + RSSR′ ⇄ RSSR + R′S· ${\rm RS}\cdot +{\rm O}_{2}\rightarrow {\rm RSO}_{2}$ . The results resembled those obtained using cysteine-cysteamine disulfide although the cha...

Reduction of disulfide bonds in peptides and proteins by dithiothreitol in liquid ammonia.

Reduction of disulfide bonds in peptides and proteins by dithiothreitol in liquid ammonia.

Action of sulfite on lysozyme

Recently an elegant method for the quantification of the number of disulfide bonds in proteins and peptides has been reported [T. W. Thannhauser, Y. Konishi, and H. A. Scheraga (1984)Anal. Biochem.138, 181–188]. The method is based on the quantification of 2-nitro-5-thiobenzoate (NTB) formed from the reaction of 2-nitro-5-thiosulfobenzoate with disulfides in the presence of excess sodium sulfite. Here it is reported that the NTB anion undergoes photochemical reaction with excess sulfite in the system, which results in the rapid disappearance of absorbance at 412 nm in the presence of light. The nonchromophoric derivative of this photochemical reaction is tentatively identified as a sulfo derivative of NTB. Based on these observations it is suggested that, for the quantification of disulfide bonds using the NTSB method, the assay should be carried out in the dark.