Measurement of sulfhydryls

Purify the modified protein from excess Ellman s reagent and reaction by-products by dialysis or gel filtration. A measurement of sulfhydryl content may be done by reading the absorbance of the modification reaction at 412nm (s = 1.36 X 104 M-1cm-1) versus a series of sulfhydryl standards treated in the same manner (e.g., cysteine). 

Modification of an introduced cysteine is both time- and concentration-dependent. It is therefore possible to measure the rate of sulfhydryl modification by applying low concentrations of MTS over short periods of time. The rate of reaction depends on the following factors (1) the permeability of the access pathway to the substituted cysteine, (2) electrostatic potentials, (3) the degree of ionization of the thiol, and (4) local steric constraints (Karlin and Akabas, 1998). The rate constant provides important information about the physicochemical environment of the introduced cysteine residue, relative to other accessible residues within... 

Nuclease is composed of 149 amino acid residues, and contains neither sulfhydryl groups nor disulfide bonds (20, 25, 26). Figure 1 shows the amino acid sequence of nuclease isolated from Staphylococcus aureus, strain V8 (20, 25-27). Physical measurements of the molecular weight of nuclease from strain V8 as well as from strain Foggi are consistent with this sequence (28). 

Ellman s reagent, 5,5 -dithio-bis-(2-nitrobenzoic acid), or DTNB, is a compound useful for the quantitative determination of sulfhydryls in solution (Ellman, 1958,1959). The disulfide of Ellman s reagent readily undergoes disulfide exchange with a free sulfhydryl to form a mixed disulfide and release of one molecule of the chromogenic substance 5-sulfido-2-nitrobenzoate, also called 5-thio-2-nitrobenzoic acid (TNB). The intense yellow color produced by the TNB anion can be measured by its absor-... 

Mezyk SP (1995) Rate constant determination for the reaction of sulfhydryl species with the hydrated electron in aqueous solution. J Phys Chem 99 13970-13975 Mezyk SP, Bartels DM (1995) Direct EPR measurement of Arrhenius parameters for the reactions of H atoms with H2O2 and D atoms with D2O2 in aqueous solution. J Chem Soc Faraday Trans 91 3127-3132... 

Most mitochondria contain an active H+/phosphate symport protein containing a sulfhydryl group essential for its translocator activity, (a) How could this translocator complicate measurements of the H+/0 ratio (as in Example 14.9), and (b) how could the presence of the sulfhydryl group be used to obviate these complications ... 

The rate of nucleophile addition to the double bond is dependent on the nature of the nucleophile as would be expected. Finley et al. (47) have measured the relative effectiveness of the sulfhydryl group of L-cysteine and the e-amino group of a-N-acetyl-L-lysine in adding to the double bond of N-acetyl dehydroalanine. At equal concentrations of the reactive species, cysteine adds some 31 times more rapidly to the double bond than does a-N-acetyl-L-lysine (47). However, when one compares these two compounds at the same pH the relative rates in favor of cysteine (pK of sulfhydryl group = 8.15) versus a-N-acetyl-L-lysine (pK of e-amino group = 10.53) are most impressive at lower pH s (Table VI). Therefore, it has been recommended that cys-... 

B. The DTNB Reaction of Metallothionein. The appearance of reactive (or "free") thiols in the 10,000 Dalton peak of rat renal cytosol, which was previously reported, was apparently inconsistent with the published properties of equine renal metallothionein—namely, that all sulfhydryl groups were present as metal mercaptide linkages. We have subsequently examined the reaction of 5,5 -dithiobis(2-nitrobenzoic) acid (DTNB, a standard agent for measurement of the SH concentrations ( )) with Zn-thionein. A bi-phasic reaction was found in which each step had DTNB dependent kr = 0.41 M", sec ... 

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