Determination and Derivatization of Protein Thiols by n-Octyldithionitrobenzoic Acid

Faulstich, Heinz; Tews, P.; Heintz, Daniela

doi:10.1006/abio.1993.1061

Cited by 39 publications

(32 citation statements)

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“…While this "traditional" 4% error of ε 412 represents a minor problem, the general reliability of Ellman's assay for protein sulfhydryls has been questioned in several studies: A number of protein sulfhydryls were seen to give an incomplete reaction with Ellman's reagent, even during prolonged assay times [12,15], due to steric or electrostatic constraints [13,16,17,18]. In one study, the kinetic problems with various proteins were overcome by using ODNB -(a less charged and more hydrophobic analogue) in place of DTNB 2- [12]. However, ODNB -is not available commercially.…”

Section: Introductionmentioning

confidence: 98%

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Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4′-dithiodipyridine

2002

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Since its introduction in 1959, Ellman's reagent (5,5'-dithio-bis(2-nitrobenzoic acid)) has been the favorite reagent for spectrophotometric measurement of protein sulfhydryls. Meanwhile however, evidence has accumulated that many protein sulfhydryls give an incomplete reaction with Ellman's reagent, even during prolonged assay times. In the present study, the kinetic problem was solved by including cystamine as a "mediator" between the protein sulfhydryl and Ellman's reagent, as previously applied in an enzymatic thiol assay [9]. As an alternative, 4,4'-dithiodipyridine (DTDP) was used in place of Ellman's reagent. Due to its small size, amphiphilic nature, and lack of charge, DTDP quickly reacts with poorly accessible protein sulfhydryls, without any catalysis by cystamine. The DTDP method and the Ellman/cystamine method were both optimized for maximal sensitivity, minimal sample consumption (detection limit 0.2 nmol mL(-1), determination limit 0.6 nmol mL(-1)), and minimal assay time (5 min). In validation experiments, both methods gave identical results and the measured sulfhydryls/protein matched the expected values. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00216-002-1347-2.

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Section: Introductionmentioning

confidence: 98%

“…Spectrophotometric thiol assays [10,11,12,13,14,15] are less sensitive, but they are rapid and simple; not even a parallel standard series must be performed when using a calibrated spectrophotometer. This explains why Ellman's assay is still a very popular method for quantification of thiols.…”

Section: Introductionmentioning

confidence: 99%

Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4′-dithiodipyridine

2002

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“…5-(n-Octyl) dithio-2-nitrobenzoate, for example, reacts more rapidly than DTNB with some protein thiol groups and is thought to reflect their being in hydrophobic environments (4)(5)(6).…”

Section: Introductionmentioning

confidence: 99%

A Kinetic Approach to Characterize the Electrostatic Environments of Thiol Groups in Proteins

Zhang

Ling

Means

1998

Bioorganic Chemistry

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“…Considering the contribution of the cross-linking part to the absorbance at 259 nm (0.4 ϫ E 338 (26)) the absorption of the adenosine part at that wavelength (⑀ 259 of ATP ϭ 16,415 M Ϫ1 cm Ϫ1 (27)) (Fig. 3a) reveals a ratio of 1:0.97 for the ATP␥S part and the cross-linking part.…”

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confidence: 99%

Identification of Contact Sites in the Actin-Thymosin β4 Complex by Distance-dependent Thiol Cross-linking

Reichert

Heintz

Echner

et al. 1996

Journal of Biological Chemistry

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Binding sites of actin and thymosin ␤4 were investigated using a set of bifunctional thiol-specific reagents, which allowed the insertion of cross-linkers of defined lengths between cysteine residues of the complexed proteins. After the cross-linkers were attached to actin specifically at either Cys 10 , Cys 374 , or to the sulfur atom of the ATP analog adenosine 5-O-(thiotriphosphate) (ATP␥S), the actin derivatives were reacted with synthetic thymosin ␤4 analogs containing a cysteine at one of the positions 6, 17, 28, 34, and 40. Immediate crosslinking as followed by UV spectroscopy was found for Cys 374 of actin and Cys 6 of thymosin ␤4, indicating that the N terminus of thymosin ␤4 is in close proximity ( 9.2 Å) to the C terminus of actin. In contrast, only insignificant reactivity was measured for all thymosin ␤4 analogs when the cross-linkers were anchored at Cys 10 of actin. A second contact site was identified by cross-linking of Cys 17 and Cys 28 in thymosin ␤4 with the ATP␥S derivative bound to actin, indicating that the hexamotif of thymosin ␤4 (positions 17-22) is in close proximity ( 9.2 Å) to the nucleotide. The importance of the amino acids 17 and 28 in thymosin ␤4 for the interaction with actin was emphasized by the finding that thymosin analogs containing cysteine in these positions exhibited strongly reduced abilities to inhibit actin polymerization.The physiological conditions within nonmuscle cells favor the assembly of actin monomers. Therefore, the pool of monomeric actin has to be maintained by complexation with small G-actin binding proteins. Particularly thymosin ␤4 (T␤4) 1 , which forms a 1:1 complex with actin monomers, is believed to be involved in preventing actin polymerization (1-6). Dissociation constants of the actin⅐T␤4 complex were found to be in the range of 0.4 -0.7 M for platelet actin and 0.7-2.0 M for muscle actin (4, 7). While complexed with T␤4, the exchange of the bound nucleotide in actin is retarded (8). In a detailed study, Vancompernolle et al. (9) have shown that binding of T␤4 to actin is mainly mediated by the hexamotif LKKTET (17-22), since loss of this sequence is paralleled by an almost complete loss of inhibitory activity. Alterations in the N-terminal part (1-16) of the peptide strongly influence the inhibitory activity of T␤4, whereas alterations in the C-terminal part (31-43) seem to be of minor importance (9). As shown by 1 H NMR spectroscopy (10, 11) T␤4 does not contain an ordered conformation in aqueous solution but tends to form an ␣-helical conformation between residues 5 and 16 (11). It has been proposed that T␤4 is likely to adopt a unique conformation upon binding actin (12).One of the binding sites of T␤4 on the actin molecule seems to be located in subdomain 1 as suggested by cross-linking studies (13,14). In order to gain more knowledge about contact sites in the actin⅐T␤4 complex, we performed a structural analysis using bifunctional thiol-specific reagents of the type alkylene-bis-[5-dithio-(2-nitrobenzoic acid)] for intermolecular crosslinki...

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Determination and Derivatization of Protein Thiols by n-Octyldithionitrobenzoic Acid

Cited by 39 publications

References 0 publications

Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4′-dithiodipyridine

Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4′-dithiodipyridine

A Kinetic Approach to Characterize the Electrostatic Environments of Thiol Groups in Proteins

Identification of Contact Sites in the Actin-Thymosin β4 Complex by Distance-dependent Thiol Cross-linking

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