Practical Protein Chemistry - A. Darbre 1989

Disulfide Bonds
Introduction

R. SCHROENLOHER, J. C. BENNETT (R. SCHROH-ENLOHER, J. C. BENNETT, University of Alabama in Birmingham, Schools of Medicine and Dentistry, Birmingham, Alabama, U.S.A.)

Disulfide Bonds are the primary type of covalent cross-linking in Proteins, connecting separate regions of a polypeptide chain or distinct chains. Intrachain disulfide bonds help maintain the conformational Stability of the folded polypeptide chain, thereby promoting the proper orientation of amino acid residues that form active sites or binding domains in Enzymes, Antibodies, and other biologically active proteins. Interchain disulfide bonds link individual subunits or chains, stabilize the "correct" folding of polypeptide chains within domains—otherwise maintained solely by non-covalent interactions—and thus play a vital role in preserving quaternary Structure.

In a series of studies on Insulin structure determination, Sanger et al. [35–37] first observed disulfide exchange during the partial Hydrolysis of Proteins in concentrated HCl at low temperatures. Detailed investigations using model systems comprising bis-2,4-dinitrophenyl-L-cystine (bis-DNP-cystine) and cystylbisglycine or oxidized Glutathione revealed that the disulfide exchange reaction proceeds in both concentrated mineral acids and mildly basic media, albeit via different mechanisms. In acidic environments, the exchange is inhibited by thiols, whereas in basic environments, thiols act as catalysts and SH-Reagents serve as inhibitors [35]. The same study established conditions for the partial hydrolysis of insulin while preserving the native positions of the disulfide bridges, thereby allowing their precise mapping within the hormone molecule. Research [41], using the interaction of cystine with oxidized glutathione as a model, examined the stability of disulfide bonds under various conditions, demonstrating that disulfide exchange is minimal at pH 2–6.5. Furthermore, proteolysis of Ribonuclease under conditions promoting disulfide exchange yielded cystine-containing Peptides encompassing only two of the protein's four disulfide bonds, which provided the first evidence that individual disulfide bonds can exhibit varying reactivities in exchange reactions. However, it is unlikely that disulfide shuffling occurs by this mechanism in native proteins, although instances of disulfide exchange with low-molecular-weight compounds are known [38, 40].

Thus, the specific strategy for determining disulfide bond locations depends on the Structural Features of the protein and the reactivity of its disulfide bridges. In any case, it is essential to prevent disulfide exchange during the course of the analysis and, consequently, to first identify an appropriate method for cleaving the native protein.

This chapter discusses standard approaches and methodologies for mapping disulfide bonds in proteins.