Reduction of disulfides

Complete Reduction of Disulfides in Protein Molecules Using DTT... 

The reduction of disulfides by 2-mercaptoethanol proceeds through a mixed disulfide intermediate. 

The reduction of disulfide bonds with trivalent phosphines has been known for sometime (Levison et ai, 1969 Ruegg and Rudingder, 1977 Kirley, 1989). Unfortunately, trialkylphos-phines generally are water-insoluble, undergo autoxidation, and are extremely odious. 

Figure 1.78 TCEP reduction of disulfides proceeds without the use of thiol compounds.

Protocol for the Complete Reduction of Disulfide Bonds within Protein Molecules... 

The following protocol for labeling proteins with 5-IAF is adapted from Gorman (1987). It is a bit unusual in that it involves reduction of disulfides with dithiothreitol (DTT) and immediate reaction with 5-IAF in excess without removal of excess reductant. The procedure can be changed to include a gel filtration step after disulfide reduction to remove excess DTT, but in any case, it should be optimized for each protein to be modified. An alternative to the use of DTT to produce sulfhydryls is thiolation with a compound that can generate free thiols upon reaction with a protein (Chapter 1, Section 4.1). 

For complete reduction of all disulfides in the presence of a denaturant, react for 16 hours at 0°C and 2 hours at room temperature. For partial reduction of disulfides, the reaction time may be reduced to 2 hours at 37°C, particularly for antibody thiol reduction, if only partial reduction of thiols in the hinge region is done. 

Konigsberg, W. (1972) Reduction of disulfide bonds in proteins with dithiothreitol. In Methods in Enzymology, (C.H.W. Hirs, and S.N. Timaseff, eds.), Vol. 25 p. 185. Academic Press, New York. 

Klafki, H. W., Pick, A. I., Pardowitz, I., Cole, T., Awni, L. A., Barnikol, H. U., Mayer, F., Kratzin, H. D., and Hilschmann, N. (1993). Reduction of disulfide bonds in an amyloidogenic Bence Jones protein leads to formation of amyloid-like fibrils in vitro. Biol. Chem. Hoppe Seyfer374, 1117-1122. 

Reduction of Disulfides and Other Oxidation States of Sulfur... 

Analogous to the reduction of disulfides, peroxides and hydroperoxides, compounds that are generally toxic are readily reduced back to alcohols by peroxidases however, in the process, other compounds including drugs can be oxidized. 

A later study (45) indicates that cucurbitin from pumpkin has a molecular weight of TT2,000 dal tons that can be electrophoretically separated into subunits of 63,000 and 56,000 dal tons. Reduction of disulfides produces polypeptides of 36,000 and 22,000 dal tons. Globulins from six cucurbits examined chromatographically (46) have molecular weights of 220,000 to 260,000 dal tons that exhibit predominantly 10.4 - 11.2 S values (about 95% of the three globulin fractions). Cucurbitin from Cucumis sativus appears a tetramer of... 

The reduction of disulfides is a reverse reaction of thiol oxidation and, under protic... 

Scheme 48 Hemithioacetals and thioacetals via reduction of disulfides in the presence of ketone.

Thompson, E. O. P., and I. J. O Donnell Quantitative reduction of disulfide bonds in proteins using high concentrations of mercapto-ethanol. Biochim. Biophys. Acta 53, 447 —449 (1961). 

It appeared probable that the reduction of disulfides to mercaptans by means of yeast would be particularly easy, since the two substances can be interconverted quite smoothly by ordinary chemical means. This prediction did not prove to be quite correct. True, Neuberg and Schwenk could reduce a disulfide by means of yeast, but they could not do so with the expected ease. Ethyl disulfide was chosen for the experiment because its boiling point (151°) differs very much from that of ethyl mercaptan (36°). Yeast which has been killed by boiling does not convert the disulfide into the mercaptan, but fermenting yeast does in view of the physiological importance of the disulfide group in cystine and glutathione, this observation is worthy of note. 

To avoid reduction of disulfide bonds, use 0.2 mg/ml tyrosin instead of 2-mercaptoethanol. 

Typically, the incorporation of carbohydrazide obtained with antibody A5B7 was about 0.5 molecules of carbohydrazide per F(ab )2 as determined by incorporation of 55Fe-labeled aldehyde 201 ESI-MS after reduction of disulfide bridges by dialysis in 10 mM DTT confirmed that the light chain was not modified by this procedure. On the other hand, the heavy-chain mass was 26 489 Da in the F(ab )2-carbohydrazide and only 26 416 in the F(ab )2. The 73 Da difference between the two heavy chains is in agreement with the 72 Da increment expected by the incorporation of one carbohydrazide group. 

Honeychurch and Ridd [97] used chronopotentiometry to study reduction of five disulfide-containing proteins (bovine serum albumin, insulin, ribonuclease A, transferrin, and trypsin) adsorbed on HMDE. All studied proteins exhibited a reduction step at —0.6 V (versus SCE), due to reduction of disulfide groups. 

Radical-anion complexes Scope of this review 91 Thermodynamic and kinetic methodologies Voltammetric methods 92 Homogeneous redox catalysis 94 Convolution analysis 98 Laser flash photolysis 102 Photoacoustic calorimetry 103 Thermochemical estimates 105 Fleduction of C—O and O O bonds 106 Reduction of ethers 107 Reduction of peroxides and endoperoxides Reduction of S—S and C—S bonds 136 Reduction of disulfides 137 Reduction of sulfides 150 Concluding remarks 157 Fleferences 160... 

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