Disulfide bond reduction, effect

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 ... 

Disulfides. The introduction of disulfide bonds can have various effects on protein stability. In T4 lyso2yme, for example, the incorporation of some disulfides increases thermal stability others reduce stability (47—49). Stabili2ation is thought to result from reduction of the conformational entropy of the unfolded state, whereas in most cases the cause of destabili2ation is the introduction of dihedral angle stress. In natural proteins, placement of a disulfide bond at most positions within the polypeptide chain would result in unacceptable constraint of the a-carbon chain. 

Figure 6.2 The trifunctional reagent sulfo-SBED reacts with amine-containing bait proteins via its NHS ester side chain. Subsequent interaction with a protein sample and exposure to UV light can cause crosslink formation with a second interacting protein. The biotin portion provides purification or labeling capability using avidin or streptavidin reagents. The disulfide bond on the NHS ester arm provides cleavability using disulfide reductants, which effectively transfers the biotin label to an unknown interacting protein.

Figure 21.5 SPDP can be used to modify both an antibody and a toxin molecule for conjugation purposes. In this case, the antibody is thiolated to contain a sulfhydryl group by modification with SPDP followed by reduction with DTT. A toxin molecule is then activated with SPDP and reacted with the thiolated antibody to effect the final conjugate through a disulfide bond.

Note Some protocols avoid a reduction step, as it can lead to disulfide bond cleavage and detrimental effects on protein activity. As an alternative to reduction, add 50 pi of 0.2 M lysine in 0.5 M 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. 

Many, but not all, proteins are sensitive to alterations in the oxidation-reduction potential of their environment. The effect is caused in part by oxidation of sulfhydryl groups or reduction of disulfide bonds. Not all proteins are equally sensitive to such alterations, but when they are, it is critical to be aware of their sensitivity. The purification or assay of some proteins can be accomplished only by providing reducing conditions (reduced glutathione, free cysteine, dithiothreitol, or mercap-toethanol) in all buffer solutions. 

The conversion of disulfide bonds to thiol groups has a much greater effect on the physical properties of wool than conversion to (S-methyl groups (Crewther, 1965b). Reduction of relatively few disulfides and the consequent small increase in thiol content give maximum extensions > 100 % and high strains at the end of the yield region. 

The predominant contribution of the folded native structure is amply illustrated by the findings that complete reduction of disulfide bonds and 5-carboxymethylation completely alters the immunochemical reactivity of protein antigens, such as ribonuclease, lysozyme, and other antigens.However, reduction of but two of the four disulfide bonds in ribonuclease had a negligible effect on the ability to precipitate with antibody to native ribonuclease. 

Renaturation. The effects of i) reduction of the disulfide bond of p-lactamase, ii) renaturation buffer pH, iii) concentration of protein, iv) GuHCl in the denaturing solution and finally v) sucrose concentration on the reversibility of the unfolding transition were investigated. 

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