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

@article{citeulike:3470098, abstract = {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.}, author = {Riener, Christian and Kada, Gerald and Gruber, Hermann}, citeulike-article-id = {3470098}, citeulike-linkout-0 = {http://dx.doi.org/10.1007/s00216-002-1347-2}, day = {1}, doi = {10.1007/s00216-002-1347-2}, journal = {Analytical and Bioanalytical Chemistry}, keywords = {cysteine, ellmans\_reagent, sh\_groups}, month = {July}, number = {4}, pages = {266--276}, posted-at = {2008-10-31 15:38:31}, priority = {2}, title = {Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4'-dithiodipyridine}, url = {http://dx.doi.org/10.1007/s00216-002-1347-2}, volume = {373}, year = {2002} }

TY - JOUR ID - citeulike:3470098 L3 - citeulike-article-id:3470098 N2 - 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. IS - 4 JF - Analytical and Bioanalytical Chemistry EP - 276 TI - Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4'-dithiodipyridine VL - 373 SP - 266 KW - cysteine KW - ellmans_reagent KW - sh_groups AU - Riener, Christian AU - Kada, Gerald AU - Gruber, Hermann PY - 2002/07/01/ UR - http://dx.doi.org/10.1007/s00216-002-1347-2 ER -