Ellman's reagent

Structural formula
General
Surname	Ellman's reagent
other names	5- (3-Carboxy-4-nitrophenyl) disulfanyl-2-nitrobenzoic acid; 5,5'-dithiobis (2-nitrobenzoic acid); Dithionitrobenzoic acid (DTNB);
Molecular formula	C 14 H 8 N 2 O 8 S 2
External identifiers / databases
EC number	200-714-4
ECHA InfoCard	100,000,650
PubChem	6254
Wikidata	Q3604488
properties
Molar mass	396.35 g mol −1
Physical state	firmly
Melting point	240–245 ° C (decomposition)
safety instructions
GHS labeling of hazardous substances Caution
H and P phrases	H: 315-319-335
	P: 261-305 + 351 + 338
	As far as possible and customary, SI units are used. Unless otherwise noted, the data given apply to standard conditions .

Ellman's reagent (5,5′-dithiobis-2-nitrobenzoic acid, DTNB) is a substance that is used in biochemistry to determine the amount of thiols in a sample.

properties

Thiols in biomolecules , especially in cysteine- containing proteins and peptides , react with DTNB, releasing 2-nitro-5-thiobenzoate (TNB ^- ), which deprotonates in neutral and basic solutions to form the yellow dye TNB ²⁻ .

The amount of released TNB ^- can be quantified photometrically at a wavelength of 412 nm . The reaction takes place in a stoichiometric ratio. The extinction coefficient in dilute solutions is 14,150 M ⁻¹ cm ⁻¹ and 13,700 M ⁻¹ cm ⁻¹ in higher salt concentrations such as 6 M guanidine hydrochloride or 8 M urea. Recrystallization of the DTNB can be carried out for reproducible results .

Alternatively, 2,4-dinitrobenzenesulfonyl fluorescein or 5- (2-aminoethyl) dithio-2-nitrobenzoic acid can be used.

synthesis

Ellman produced DTNB in several steps by oxidizing 2-nitro-5-chlorobenzaldehyde to the carboxyl derivative , by generating the thiol with sodium sulfide and by oxidizing the thiol groups to disulfide bonds with iodine .

use

Ellman's reagent can be used to determine glutathione and thiol groups in proteins in purified solutions or in biological samples such as blood . The reaction with free thiol groups produces yellowish p-nitrophenyl, which can be detected spectroscopically at 412 nm.

Ellman's reagent can also be used to rapidly and specifically form inter- and intramolecular disulfide bridges between cysteines of peptides over a wide range of pH values. In solid phase synthesis, large yields are achieved with little oligomerization. The reagent binds to the solid phase on two sides. A bond is made to a thiol group. A second thiol group displaces the reagent and is thus linked to the first to form a disulfide. The thiol groups on Ellman's reagent are reduced in the process.

history

DTNB was first manufactured by George L. Ellman in 1958.

Web links

Quantitation of sulfhydryls DTNB, Ellman's reagent (PDF)

Individual evidence

↑ ^a ^b ^c data sheet 5,5′-dithiobis (2-nitrobenzoic acid) from Sigma-Aldrich , accessed on July 17, 2016 ( PDF ).
↑ ^a ^b ^c G. Ellman: A colorimetric method for determining low concentrations of mercaptans . In: Arch. Biochem. Biophys . 74, No. 2, 1958, pp. 443-450. doi : 10.1016 / 0003-9861 (58) 90014-6 .
^ ^A ^b Ellman GL: Tissue sulfhydryl groups . In: Arch. Biochem. Biophys. . 82, No. 1, 1959, pp. 70-77. doi : 10.1016 / 0003-9861 (59) 90090-6 . PMID 13650640 .
↑ Collier HB: Letter: A note on the molar absorptivity of reduced Ellman's reagent, 3-carboxylato-4-nitrothiophenolate . In: Anal. Biochem. . 56, No. 1, 1973, pp. 310-1. doi : 10.1016 / 0003-2697 (73) 90196-6 . PMID 4764694 .
↑ ^a ^b P. W. Riddles, RL Blakeley, B. Zerner: Reassessment of Ellman's reagent. In: Methods in enzymology. Volume 91, 1983, ISSN 0076-6879 , pp. 49-60, doi : 10.1016 / S0076-6879 (83) 91010-8 , PMID 6855597 .
↑ H. Maeda, H. Matsuno, M. Ushida, K. Katayama, K. Saeki, N. Itoh: 2,4-Dinitrobenzenesulfonyl fluoresceins as fluorescent alternatives to Ellman's reagent in thiol-quantification enzyme assays. In: Angewandte Chemie (International ed. In English). Volume 44, Number 19, May 2005, pp. 2922-2925, ISSN 1433-7851 . doi : 10.1002 / anie.200500114 . PMID 15818626 .
↑ J. Zhu, I. Dhimitruka, D. Pei: 5- (2-Aminoethyl) dithio-2-nitrobenzoate as a more base-stable alternative to Ellman's reagent. In: Organic letters. Volume 6, Number 21, October 2004, pp. 3809-3812, ISSN 1523-7060 , doi : 10.1021 / ol048404 + , PMID 15469355 .
↑ Sedlak J, Lindsay RH: Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent . In: Anal. Biochem. . 25, No. 1, 1968, pp. 192-205. doi : 10.1016 / 0003-2697 (68) 90092-4 . PMID 4973948 .
↑ Riener CK, Kada G, Gruber HJ: Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4'-dithiodipyridine . In: Anal Bioanal Chem . 373, No. 4-5, 2002, pp. 266-76. doi : 10.1007 / s00216-002-1347-2 . PMID 12110978 .
^ RJ Simpson: Estimation of Free Thiols and Disulfide Bonds Using Ellman's Reagent. In: CSH protocols. Volume 2008, 2008, pdb.prot4699, PMID 21356901 .
↑ Annis I, Chen L, Barany G: Novel Solid-Phase Reagents for Facile Formation of Intramolecular Disulfide Bridges in Peptides under Mild Conditions . In: J. Am. Soc. . 120, No. 29, 1998, pp. 7226-7238. doi : 10.1021 / ja981111p .

[sigma-1] ta sheet 5,5′-dithiobis (2-nitrobenzoic acid) from Sigma-Aldrich , accessed on July 17, 2016 ( PDF ).

[ellman1-2] G. Ellman: A colorimetric method for determining low concentrations of mercaptans . In: Arch. Biochem. Biophys . 74, No. 2, 1958, pp. 443-450. doi : 10.1016 / 0003-9861 (58) 90014-6 .

[ellman2-3] A ^b Ellman GL: Tissue sulfhydryl groups . In: Arch. Biochem. Biophys. . 82, No. 1, 1959, pp. 70-77. doi : 10.1016 / 0003-9861 (59) 90090-6 . PMID 13650640 .

[4] Collier HB: Letter: A note on the molar absorptivity of reduced Ellman's reagent, 3-carboxylato-4-nitrothiophenolate . In: Anal. Biochem. . 56, No. 1, 1973, pp. 310-1. doi : 10.1016 / 0003-2697 (73) 90196-6 . PMID 4764694 .

[Riddles-5] P. W. Riddles, RL Blakeley, B. Zerner: Reassessment of Ellman's reagent. In: Methods in enzymology. Volume 91, 1983, ISSN 0076-6879 , pp. 49-60, doi : 10.1016 / S0076-6879 (83) 91010-8 , PMID 6855597 .

[6] H. Maeda, H. Matsuno, M. Ushida, K. Katayama, K. Saeki, N. Itoh: 2,4-Dinitrobenzenesulfonyl fluoresceins as fluorescent alternatives to Ellman's reagent in thiol-quantification enzyme assays. In: Angewandte Chemie (International ed. In English). Volume 44, Number 19, May 2005, pp. 2922-2925, ISSN 1433-7851 . doi : 10.1002 / anie.200500114 . PMID 15818626 .

[7] J. Zhu, I. Dhimitruka, D. Pei: 5- (2-Aminoethyl) dithio-2-nitrobenzoate as a more base-stable alternative to Ellman's reagent. In: Organic letters. Volume 6, Number 21, October 2004, pp. 3809-3812, ISSN 1523-7060 , doi : 10.1021 / ol048404 + , PMID 15469355 .

[8] Sedlak J, Lindsay RH: Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent . In: Anal. Biochem. . 25, No. 1, 1968, pp. 192-205. doi : 10.1016 / 0003-2697 (68) 90092-4 . PMID 4973948 .

[Riener-9] Riener CK, Kada G, Gruber HJ: Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4'-dithiodipyridine . In: Anal Bioanal Chem . 373, No. 4-5, 2002, pp. 266-76. doi : 10.1007 / s00216-002-1347-2 . PMID 12110978 .

[10] RJ Simpson: Estimation of Free Thiols and Disulfide Bonds Using Ellman's Reagent. In: CSH protocols. Volume 2008, 2008, pdb.prot4699, PMID 21356901 .

[11] Annis I, Chen L, Barany G: Novel Solid-Phase Reagents for Facile Formation of Intramolecular Disulfide Bridges in Peptides under Mild Conditions . In: J. Am. Soc. . 120, No. 29, 1998, pp. 7226-7238. doi : 10.1021 / ja981111p .