Disulfide bond reduction and

Mekalanos JJ, Collier RJ, Romig WR (1979) Enzymatic activity of cholera toxin II. Relationships to proteolytic processing, disulfide bond reduction, and subunit composition. In J. Biol. Chem. 254 5855-5861. 

Fig. 6.22. Folate-FRET sensor structure and its application to measure disulfide bond reduction in endosomes. The molecule contains the folate moiety which is recognized by the folate receptor situated at the plasma membrane. This recognition leads to endocytosis and after some time to cleavage of the probe. 

Note Some protocols do not call for a reduction step. The addition of borohydride at this level may result in disulfide bond cleavage and loss of protein activity in some cases. As an alternative to reduction, add 50pi of 0.2M lysine in 0.5M sodium carbonate, pH 9.5 to each ml of the conjugation reaction to block excess reactive sites. Block for 2 hours at room temperature. Other amine-containing small molecules may be substituted for lysine—such as glycine, Tris buffer, or ethanolamine. 

Cline DJ, et al. New water-soluble phosphines as reductants of peptide and protein disulfide bonds reactivity and membrane permeability. Biochemistry 2004 43 15195. 

THE CELLULAR FUNCTION OF ACROSIN AS A SPERM LYSIN. Acrosin fulfills most of the above listed criteria for a sperm lysin. It has been localized onto both the inner and outer acrosomal membranes by histochemical and immunochemical localization procedures (60.61). and is at the correct place to function in fertilization. Complete ZP dissolution by homologous acrosins, the second lysin criterion, is not observed with all species. With boar acrosin, dissolution of the porcine ZP was never visually observed, as was true with sheep ZP and ram acrosin (62). To visualize the effect of acrosin on the ZP, electrophoretic or other separation procedures that employed denaturants and disulfide bond reduction were required (7.37.38). Complete dissolution of the ZP does not occur in normal fertilization, since the ZP is needed for the continued development of the embryo ... 

PDI disulfide bond formation and isomerization, folding dehydro ascorb ate reduction dehydro ascorb ate, GSH 10,11,22,23, 44,46,54... 

Disulfide (-S-S-) bonds play an important role in the structure and function of proteins. IgG molecules are comprised of two heavy and two light chains linked by interchain disulfide bonds. In addition, intrachain disulfide bonds are also present in IgGs. Alkyl thiols have been used to demonstrate the structural and functional role played by disulfide bonds. Reduction of IgGs with alkyl thiols under denaturing conditions results in separation of light and heavy chains. 

Fig. 17.7. Engineered protein designed for mechanochemistry studies, (a) A pair of cysteine residues introduced into the 127 protein (positions 32 and 75 suifur atoms as spheres) spontaneously form a buried disuifide bond. (b). In response to an unfolding force, the protein extends right up to the disuifide bond. Unfoiding exposes the disulfide bond to the solution, (c) Then, a nucieophiie such as DTT can initiate a Sn2 reaction, leading to the reduction of the disuifide bond and the concomitant extension of the amino acids that were trapped behind the disuifide bond. This sequence of events nnambiguousiy identifies individual disulfide bond reduction events, allowing for the stndy of a pulling force on a Sn2 chemicai reaction...

As for reduction processes, C02 free radicals were shown to react specifically with disulfide bonds (122). They were extensively used to study the redox properties of disulfide bonds, thiyl and disulfide free radicals in proteins. This is discussed in paragraph 5. However, they do react with thiol functions also (37). For proteins containing a prosthetic group, the reduction concerns also oxidized valencies of metals and flavins. Flavin adenine dinucleotide (FAD) or Flavin Mononucleotide (FMN). The proportion of reduced disulfide/reduced prosthetic group varies considerably with the protein. For instance, lipoamide dehydrogenase contains one disulfide bond close to a flavin (FAD). Free radicals can reduce only the flavin, although both are in the active site (123). In chicken egg white riboflavin binding protein, competitive formation of both disulfide and semireduced flavin is observed (124). 

Kuwajima K, Ikeguchi M, Sugawara T, et al. (1990). Kinetics of disulfide bond reduction in a-lactalbumin by dithiothreitol and molecular basis of superreactivity of the cys6-cysl20 disulfide bond. Biochem. 29 8240-8249. 

Perreault, S. D., Wolff, R. A., and Zirkin, B. R. (1984). The role of disulfide bond reduction during mammalian sperm nuclear decondensation in vivo. Dev. Biol 101, 160-167. 

Compare the oxidized and reduced chemokine spectra to confirm that disulfide bond reduction caused the NMR signals to collapse (Fig. 6, bottom). 

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