Thiol redox buffer

As well as its role as a general thiol redox buffer, GSH acts as a co-factor for a variety of enzymes, including GSH-dependent peroxidases, glutathione-S-transferases (GST), glutaredoxins and glyoxalase. 

GSH synthase, a tripeptide (glutamylcysteinylglycine), is not only a water-soluble antioxidant, but is also part of a redox buffer (Smith et al., 1996). It is found in all cells and is used for a multiplicity of cellular functions, such as protein and prostaglandin synthesis, detoxification, etc. Cytosolic concentrations of GSH range from 1 to 11 mm (Smith et al., 1996) and are 100-1000 times greater than the extracellular levels. Many proteins contain sulfhydryl groups because of their cysteine content. The content of thiols in proteins is greater than that of the pool of GSH (Torchinsky, 1981). 

Dissolve the previously reduced and purified poly(thiol) peptide in the above redox buffer to a concentration of 0.05-0.1 mg/ml (= 0.05-0.1 mM). 

The reactive site of the cysteinyl residue is the thiol group, which is deprotonated at alkaline pH (pXa around 8.5). The residue under oxidizing conditions (and neutral to alkaline pH) is able to react with a similar residue under formation of a disulfide bond. Many proteins are stabilized by intramolecular disulfide bonds (e.g., insulin, growth hormone, lGF-1), but intermolecular bonds may also result from the reaction under formation of aggregates. In order to avoid unintended disulfide bond formation/cleavage, the redox potential of the solution must be monitored and controlled. In practice, aqueous buffers contain micromolar amounts of dissolved oxygen assuring a redox potential of 200-600 mV, which is sufficient to maintain the intramolecular disulfide bonds. Proteins with free cysteines may... 

Fig. 6. (A) The electrochemical detection of hybridization on Au electrode surface. Probe oligonucleotides with 5 -thiol are incubated on the Au surface for 12h. Strong covalent bond between thiols and Au surface results in the formation of the probe-modified electrode (A1), noncomplementary oligonucleotides are incubated with the probe-modified electrode (A2) however, no hybridization takes place between these two strands (A3), the redox label, Hoechst 33258, is incubated with the probe-modified-electrode (A4), since the redox label cannot intercalate with the single-strand probe oligonucleotides on the surface, a small electrochemical current response is obtained (A5). The current intensity at the peak potential (-0.6V) is recorded relative to the background response of the blank buffer solution (dashed grey line). (B) The electrochemical detection of hybridization on Au electrode surface. Probe oligonucleotides with 5 -thiol are incubated on the Au surface for -12 h for the preparation of the probe-modified electrode (B1), target oligonucleotides are...

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