Sulfhydryl estimation

Add iodoacetate to a concentration of 50mM in the reaction solution. Alternatively, add a quantity of iodoacetate representing a 10-fold molar excess relative to the number of —SH groups present. An estimation of the sulfhydryl content in the protein to be modified can be accomplished by performing an Ellman s assay (Chapter 1, Section 4.1). Readjust the pH if necessary. To aid in adding a small quantity of iodoacetic acid to the reaction, a concentrated stock solution may be made in the reaction buffer, the pH re-adjusted, and an aliquot added to the protein solution to give the desired concentration. 

Byler, D. M., Gosser, D. K., and Susi, H. (1983). Spectroscopic estimation of the extent of S-nitrosothiol formation by nitrite action on sulfhydryl groups. J. Agric. FoodChem. 31, 523-527. 

At appreciable ionic strength these microscopic constants should be written in terms of activities rather than concentrations. The constants are normally estimated by spectral means. There are three microscopic ionization constants for the loss of the first proton, six for the loss of the second, and three for the loss of the last. In Table 3-2 the subscripts 1, 2, and 3 denote the carboxyl, sulfhydryl, and ammonium protons thus 32 is the microscopic constant for the ion formed by loss of the sulfhydryl proton from the species that has already lost the ammonium proton. 

Fig. 12.—Interaction of /S-Galactoaidaae of E. coU, ML 309, with p-(Chloromer-curi)benzoate at 5 (XXX), 20 (OOO), and 40° (AAA). [Sulfhydryl groups were estimated by Boyer s method (p-(chloromercuri)benzoate 5 X 10 M, enzyme 250 pg./inl.). The activity was measured with phenyl /8-u-galactoside (1 X 10" M in 2-amino-2-(hydroxymethyl)-l,3-propanediol-HCl buffer, pH 7.6). A = Enzyme activity in the presence of p-(chloromercuri)benzoate A = enzyme activity in the absence of p-(chloromercuri)benzoate.j...

Myosin. I. Sulfhydryl Groups as Estimated by Porphyrindin Titration. 

OPDM). One of two maleimide residues of OPDM, added in excess (10 pM) to the protein, reacts with the thiol group, whereas the other remains inactive. The number of maleimide-reacted thiol groups in the protein can be estimated by the determination of the number of unmodified sulfhydryl groups. Subsequently, the maleimide-deri-vatized protein is conjugated with BGase. 

Sulfhydryl content can be determined directly by using any thiol assay, typically by using Ellman s reagent, although other more sensitive procedures exist (47), or indirectly by release of some reporter molecule on deprotection of crosslinkers. For example, many reports use the absorbance of 2-thiopyridone at 343 nm after treatment of pyridyldithio-modified proteins with DTT. Extreme caution should be used when interpreting results from these indirect methods since in our experience treatment of antibodies with DTT, even under mild conditions, results in subtle changes in the baseline absorbance at 343 nm and consequent erroneous estimates of thiol content. 

The reduction of the keto groups to enol groups occurs with many reducing compounds. Of particular interest are the various sulfhydryl compounds (H2S, GSH, homocysteine used to convert dehydroascorhic acid to ascorbic acid for analysis by an oxidation method. Similar compounds can potentially reduce dehydroascorhic acid in biological systems. The redox potential of GSH is now believed to be considerably lower than was once thought (Eo at pH 7, 30° C = —0.32) (B28), considerably below that of ascorbic acid (-(-0.058). This in part accounts for the inefficiency of sulfhydryl reductions of dehydroascorhic acid. Mapson (M6) estimates that Ae half-time of reduction of dehydro-ascorbic acid by GSH is about 15 minutes under physiological conditions of pH, temperature and concentrations. 

No disulfide bridges have been detected in any GDH. Estimates of the sulfhydryl content of the bovine enzyme have given values of 7.2 by titration in guanidine hydrochloride with o-iodosobenzoate 158) and... 

Whereas a carboxyl break was readily observed witti alkali potentiometry, ttie sulfhydryl break could not be detected (17). Therefore, the plCg of the sulfhydryl in captopril (PIC2) was not estimated by classical potentiometry. It was, however, estimated at 9.8 (pJ 2) by Ondetti (19) and Weiss (30) using sulfliydryl u.v. shifts to higher wavelengths with increase in pH. The method utilized was adapted from Benesch and Benesch (31)."... 

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