title: Molar absorption coefficients for the reduced Ellman reagent: reassessment.
abstract: The Ellman method for assaying thiols is based on the reaction of thiols with the chromogenic DTNB (5,5'-dithiobis-2-nitrobenzoate) whereby formation of the yellow dianion of 5-thio-2-nitrobenzoic acid (TNB) is measured. The TNB molar absorption coefficient, 13.6 x 10(3)M(-1)cm(-1), as published by Ellman in 1959 has been almost universally used until now. Over the years, however, slightly different values have been published, and it has further been shown that TNB reveals thermochromic properties. This should be taken into account when the Ellman method is used for determination of enzyme activities, such as in cholinesterase assays. Our data show that the absorbance spectra of TNB are shifted to longer wavelengths when temperature increases, while absorbance maxima decrease. Our recommended molar absorption coefficients at 412 nm are 14.15 x 10(3)M(-1)cm(-1) at 25 degrees C and 13.8 x 10(3)M(-1)cm(-1) at 37 degrees C (0.1M phosphate buffer, pH 7.4). Molar absorption coefficients for other temperatures and wavelengths are included in the paper.
authors.affiliation: Walther-Straub-Institute of Pharmacology and Toxicology, University of Munich, Nussbaumstrasse 26, Germany. peter.eyer@lrz.uni-muenchen.de
authors.forename: Peter
authors.initials: P
authors.lastname: Eyer
authors.forename: Franz
authors.initials: F
authors.lastname: Worek
authors.forename: Daniela
authors.initials: D
authors.lastname: Kiderlen
authors.forename: Goran
authors.initials: G
authors.lastname: Sinko
authors.forename: Anita
authors.initials: A
authors.lastname: Stuglin
authors.forename: Vera
authors.initials: V
authors.lastname: Simeon-Rudolf
authors.forename: Elsa
authors.initials: E
authors.lastname: Reiner
mesh_terms: D000042:Absorption; D004228:Dithionitrobenzoic Acid / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D013696:Temperature
chemical_list: D004228:Dithionitrobenzoic Acid