Sulfhydryl content

Jaeger RJ, Cote IL, Rogers AE, et al. 1984. Acute toxicity of acrylonitrile Effect of diet on tissue nonprotein sulfhydryl content and distribution of 1- -acrylonitrile or its metabolites. J Am Coll Toxicol 3 93-102. 

The deacetylated protein should be used immediately to prevent loss of sulfhydryl content through disulfide formation. The degree of—SH modification may be determined by performing an Ellman s assay (Section 4.1, this chapter). 

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. 

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). 

Chang has made observations on the polysomes of pinto bean leaves exposed to ozone (at 0.35 ppm for 20-50 min). He found that the chloroplast polysomes were more susceptible to oxidation than was the cytoplasmic ribosomes. The sulfhydryl content of the chloroplast ribosomes was also much more susceptible to oxidation than was that of the cytoplasmic ribosomes. Finally, it was found that the effects of ozone on ribosome composition could be reproduced by p-mer-curicbenzoate. Chang s results imply that either ozone itself or a product of ozone oxidation passes from the cytoplasmic membrane to the interior of the chloroplast before having its effect. These results connect with a number of papers on the oxidation of sulfhydryl compounds by ozone. Tomlinson and Rich have reported decreases in leaf sulfhydryl groups after ozone exposure (at 1 ppm for 30-60 min). 

A number of investigators have found that reagents that react with sulfhydryl groups can reproduce the symptoms of ozone damage. Dass and Weaver reported this effect in white bean. Tomlinson and Rich reported the same effect in tobacco, and Rich and Tomlinson found that conidiophores of Altemaria solani became more susceptible to ozone if they were pretreated with iodoacetamide. It appears that effects on sulfhydryl content may be quite early events in the toxic reactions initiated by ozone. One should remember, however, that symptoms of ozone and PAN injury are quite different, and one cannot explain the effects of both pollutants as sulfhydryl oxidizers. 

In our first experiments (1) we subjected bean, spinach (Spinacia oleracea L.) and tobacco leaves to ozone at 1 ppm for 30 to 60 min. At this high concentration of ozone, the sulfhydryl content of the leaves was diminished 15 to 25% (Table I). There was little difference between the sulfhydryl content of ozone-resistant and ozone-susceptible tobacco either before or after ozonation. 

In later experiments 2)9 beans were subjected to a milder, longer exposure to ozone (25 pphm for 3 hr). This treatment did not diminish the sulfhydryl content appreciably, even though the ozonated leaves showed injury 18 hr later. However, we were able to detect newly produced disulfides (Table III). We concluded that ozonation changes proteins sufficiently to expose and oxidize additional sulfhydryl groups. 

NT059 Singh, A., and S. P. Singh. Modulatory potential of smokeless tobacco on the garlic, mace or black mustard-altered hepatic detoxication system enzymes, sulfhydryl content and lipid peroxidation in murine system. Cancer Lett... 

Kreiling, R., Laib. R.J. Bolt, H.M. (1988) Depletion of hepatic non-protein sulfhydryl content during exposure of rats and mice to butadiene. Toxicol. Lett., 41, 209-214... 

Forkert, P.G. Moussa, M. (1993) Temporal effects of 1,1-dichloroethylene on nonprotein sulfhydryl content in murine lung and liver. DrugMetab. Dispos., 21, 770-776... 

Agarwal DK, Agarwal S, Seth PK. 1982a. Effect of di(2-ethylhexyl)phthalate on drug metabolism, lipid peroxidation, and sulfhydryl content of rat liver. DrugMetab Dispos 10 77-80. 

Singer EJ, Wegmann PC, Lehman MD, Christensen MS, Vinson LJ (1971) Barrier development, ultrastructure, and sulfhydryl content of the fetal epidermis. J Soc Cosmet Chem, 22 119-137. 

Figure 17. The effect of disulfide bond modification of turkey ovomucoid by alkali on inhibitory activity against trypsin (T), a-chymotrypsin, (C), and subtilisin (S). Turkey ovomucoid (O.lOmM) was treated with alkali (lOOmM NaOH) at 23°C. Sulfhydryl content (moles per mole of protein)...

The effect of administration of benzene, 0.5 mL/kg intraperitoneally or 1 mL/kg subcutaneously, to male and female rats for 10 consecutive days on free sulfhydryl content and lipid peroxidation in the liver was investigated (Ahmad et al. 1994). The free sulfhydryl content of the liver showed a significant decrease in... 

It is well known that lipid peroxidation, DNA singlestrand breaks, and other forms of DNA damage occur in response to oxidative stress (Ames et al, 1982). Depletion of reduced glutathione also commonly precedes or accompanies lipid peroxidation and oxidative stress (Muldoon and Stohs, 1991 Omar et al, 1990). In an in vivo study, the effects of ricin administered orally on hepatic lipid peroxidation, nonprotein sulfhydryl content, and DNA singlestrand breaks were assessed in mice (Muldoon et al, 1992). The incidence of hepatic DNA damage increased 2.9-, 2.8-, and 2.4-fold relative to control values at 24, 36, and 48 h post-treatment with ricin, respectively. Hepatic nonprotein sulfhydryl concentration decreased significantly from 51 to 65% to control values at 24, 36, and 48 h post-treatment (Figures 25.2 and 25.3). 

The Effects of Ricin on Hepatic Nonprotein Sulfhydryl Content... 

The nature of the active site in beta-amylase is not unambiguously known for enzymes from different sources. Early experiments on purified barley and on malted barley first indicated, from studies of the modification of the enzyme with nitrous acid and ketene, that free tyrosine and sulfhydryl groups are essential for activity, whereas free a-amino groups are not. The importance of the sulfhydryl groups was emphasized by the partial recovery of activity of the modified or oxidized enzyme (that is, treated with nitrous acid, iodine, phenyl mercuribenzoate, ferricyanide, and cupric ions) when it was treated with hydrogen sulfide or cysteine. Barley feeto-amylase (not highly purified) has been reported to contain 12—15 sulfhydryl groups per molecule by titration with p-chloromercuribenzoate, and the loss of free sulfhydryl content by treatment with L-ascorbic acid in the presence of cupric ions was found to be directly related to the loss of activity. 

The paper by Janatova et al. (1968) should be consulted for a careful study of the optimum conditions for the determination of the sulfhydryl content of native proteins by titration with DTNB. 

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