Disulfide bond cleavage

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. 

The first step, protein purification, is discussed in chapter 5. Once the protein is pure, sequence analysis can begin, with cleavage of the disulfide bonds. Cleavage is achieved by oxidizing the disulfide linkages with performic acid (fig. 3.17). Sometimes this step results in the production of two or more polypeptide chains, in which case the individual chains must be separated. 

Brody, 1. (1997), Multistep dcnaiuration and hierarchy of disulfide bond cleavage of a monoclonal antibody. AiwJ. Biochem. 247,247-256. 

Kalapathy, U. N.S. Hettiarachchy D.J. Myers K.C. Rhee. Alkali modification of soy proteins effects of salts and disulfide bond cleavage on adhesion and viscosity. J. Am. Oil Chem. Soc. 1996, 73, 1063-1066. 

The actual oxidizing species present depends on pH and is either chlorine or hypochlorous acid (HOCl). Apparently, the hypochlorous acid is the more active species on hair given that degradation is greater at lower pH. Although the chemistry of these interactions was not examined, one would expect disulfide bond cleavage and peptide bond fission similar to the effects shown for the reaction of chlorine and wool fiber [42]. 

Hofbauer F, Frank I (2010) Disulfide bond cleavage a redox reaction without electron transfer. ChemEur J 16 5097-5101... 

Dopieralski P, Ribas-Aiino J, Anjukandi P, Krapicka M, Kiss J, Marx D (2013) The Janusfaced role of external forces in mechanochemical disulfide bond cleavage. Nat Chtan 5 ... 

Sawicka, A. Skurski, R Hudgins, R.R. Simons, J. Model calculations relevant to disulfide bond cleavage via electron capture influenced by positively charged groups. J. Phys. Chem. B 2003,107, 13505-13511. 

Nielsen HC, Beckwith AC, Wall JS. Effect of disulfide-bond cleavage on wheat gliadin fractions obtained by gel filtration. Cereal Chem 1968 45 37-47. 

A more subtle problem is presented by proteins that associate or polymerize, noncovalently or covalently, but remain soluble. One example for zein was noted before (Fig. 5) a broad envelope underneath well-resolved peaks may suggest association or polymerization. Similarly, Huebner and Bietz [22] showed that medium-MW oligomeric wheat glutenin molecules eluted upon RP- HPLC as a broad peak but resolved as sharp peaks after disulfide bond cleavage. 

Chemical modification by disulfide bond cleavage can markedly alter thermal stabilities or other properties of the inhibitors, since disulfide crosslinks help maintain the active conformations of many proteinase inhibitors (Laskowski and Sealock, 1971 Tschesche, 1974 Weder, 1981). Loss of thermal stability resulting from reductive alkylation of disulfide bonds has been observed for BPTI by H-NMR (WQthrich et al., 1980), for proteinase inhibitor I from potato by immunological assay (Plunkett and Ryan, 1980), and for STI, LBI, and chicken ovomucoid by DSC (Friedman et al., 1982c). 

Mrhalca, R. van der Burgt, Y.E.M. Heck, A.J.R. Heeren, R.M.A. Disulfide Bond Cleavages Observed in SORI-CID of Three Nonapeptides Complexed With Divalent Transition-Metal Cations. J. Mass Spectrom. 2007,42,450-458. 

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