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Figure 9
The principle of and representative results from the cysteine-accessibility assay using Ellman's reagent. (a) DTNB reacts with accessible thiols such as cysteine residues in the protein to generate NTB2−, which absorbs at 412 nm. Mercury binds to accessible cysteine residues by forming a covalent Hg—S bond, thus preventing the production of NTB2− upon the addition of DTNB. (b) Progress curve of DTNB reacting with CM46 with (dashed line) or without (solid line) pre-labelling with EMP. A control sample without mercury and protein is included (dotted line). Protein was used at 30 µM. EMP, when present, was incubated with protein at a 3:1 molar ratio at 20°C for 30 min prior to the addition of DTNB. DTNB was added to a concentration of 0.2 mM to initiate the reaction. (c) Progress curve of the Ellmann reaction with CM43 (30 µM) without added mercury (solid line) and upon incubation with EMP (dotted line) or with Hg(O2CCH3)2 (mercuric acetate; dashed line). An increase of 0.26 in A412 is expected for the complete reaction of a single thiol with DTNB under the conditions indicated. The insets show the location of the single cysteine residues in the solved protein.

Journal logoBIOLOGICAL
ISSN: 1399-0047
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