DTNB (Ellman's Reagent)

Ellman's Reagent prevents dephosphorylation of histones during isolation of mitotic chromosomes

Chromosome Res 2022 Dec;30(4):351-359.36399199 10.1007/s10577-022-09709-1

Histones H1 and H3 are highly phosphorylated in mitotic HeLa cells but are rapidly dephosphorylated by endogenous protein phosphatases during the isolation of metaphase chromosomes. We show that this dephosphorylation can be prevented by including the sulfhydryl reagent 5,5'-dithiobis-(2-nitrobenzoate) (Ellman's Reagent, or DTNB) in the isolation buffer. The minimal amount of DTNB required is approximately stoichiometric with the number of sulfhydryl groups in the lysate. Inhibition of the protein phosphatases can subsequently be reversed by treatment with dithiothreitol or 2-mercaptoethanol. DTNB is compatible with the isolation of either metaphase chromosome clusters or individual metaphase chromosomes. It should be useful in investigations of the structure and biochemistry of chromatin and chromosomes and in the study of possible functions for mitotic histone phosphorylation.

Ellman's Reagent in promoting crystallization and structure determination of Anabaena CcbP

Acta Crystallogr Sect F Struct Biol Cryst Commun 2012 Nov 1;68(Pt 11):1409-14.23143261 PMC3515393

Obtaining crystals presented a bottleneck in the structural study of Anabaena cyanobacterial Ca2+-binding protein (CcbP). In this report, the promoting effect of Ellman's Reagent [5,5'-dithiobis(2-nitrobenzoic acid); DTNB] on the crystallization of CcbP is described. CcbP contains one free cysteine. A quick and simple oxidation reaction with DTNB blocked the free cysteine in purified CcbP and generated a homogenous monomeric protein for crystallization. The crystal structure of DTNB-modified CcbP was determined by the single-wavelength anomalous diffraction method. Structure analysis indicated that DTNB modification facilitated crystallization of CcbP by inducing polar interactions in the crystal lattice. DTNB-mediated cysteine modification was demonstrated to have little effect on the overall structure and the Ca2+ binding of CcbP. Thus, DTNB modification may provide a simple and general approach for protein modification to improve the success of crystallization screening.

Reassessment of Ellman's Reagent

Methods Enzymol 1983;91:49-60.6855597 10.1016/s0076-6879(83)91010-8

Ellman's Reagent: 5,5'-dithiobis(2-nitrobenzoic acid)--a reexamination

Anal Biochem 1979 Apr 1;94(1):75-81.37780 10.1016/0003-2697(79)90792-9

Thiol-disulfide exchange reactions occurring at modified bovine serum albumin detected using Ellman's Reagent (5, 5'-dithiobis (2-itrobenzoic acid)

Pak J Pharm Sci 2020 Nov;33(6(Supplementary)):2767-2772.33879435

Bovine serum albumin (BSA) is usually employed as a model protein because of being homologous with human serum albumin. Cysteine-34 of BSA has been oxidised with Ellman's Reagent to produce BSA labelled with an Ellman's moiety (BSA-SE). The BSA-SE was then reacted with glutathione, N-acetylcysteine and D-penicillamine (D-pen). The two were able to release the Ellman's moiety bound at cysteine-34 while D-pen did not. Albumin labeled using Ellman's Reagent was used to demonstrate the cleavage of a protein mixed disulphide. The kinetics of thiol disulfide interchange reactions involving formation of a chromophoric thiolate were determined by UV-visible spectroscopy. The reaction of thiolates with excess Ellman's Reagent is used for quantitative estimation of thiol by measuring the absorption at 位, 412 nm. The disulfide exchange reactions occurring at Cys-34 of BSA was determined and the reduction of oxidized Cys-34 was studied in order to understand the reverse reaction. Spectroscopic evidence suggested that glutathione and N-acetylcysteine remove the label and produce BSA in a disulfide form. In contrast, D-pen reaction returned BSA to its thiolate form via mediation. It was observed that thio-disulfide exchange occurred at cysteine-34 labelled with Ellman's moiety. The implications to the redox status of plasma are discussed.