Practical Protein Chemistry - A. Darbre 1989

Disulfide Bonds
Schematic Diagram of the Analysis

The fundamental workflow for identifying interconnected half-cystine residues has remained essentially unchanged since it was first devised by Ryle during his studies on bovine Insulin [36]. This Procedure involves the following stages:

1) partial Hydrolysis of the protein under conditions that prevent disulfide bond rearrangement;

2) fractionation of the resulting mixture to isolate cystine-containing Peptides, also under conditions that preclude disulfide exchange;

3) Cleavage of cystine-containing peptides into cysteinyl peptides;

4) isolation of individual cysteinyl peptides;

5) identification of the resulting peptides.

A more detailed stepwise analysis plan is shown in Fig. 4.1.

For the unambiguous Identification of disulfide bond positions, it is crucial that each cystine-containing peptide includes only a single disulfide bridge. It is first necessary to determine the number and positions of Cysteine residues within the polypeptide chain; therefore, this analysis is undertaken only after the complete Amino Acid Sequence of the polypeptide chain(s) under investigation has been established. The number of Disulfide Bonds can be determined using various Methods. To this end, alongside establishing the Primary Structure, the PHYSICOCHEMICAL PROPERTIES OF the protein are investigated. Data regarding intra- and interchain disulfide bonds can be obtained through physicochemical techniques. Let us illustrate this with the following example [13, 19].

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FIG. 4.1. General workflow for determining the positions of disulfide bonds in purified Proteins AND PEPTIDES.

Following mild reduction with dithiothreitol, a human IgG immunoglobulin molecule incorporates 8 moles of [¹⁴C]iodoacetamide, whereas the native protein incorporates only 0.5 moles of this reagent. Since the reduction is carried out under conditions ensuring complete dissociation of The polypeptide chains, this indicates that the immunoglobulin molecule contains no more than four interchain disulfide bridges. Consequently, the remaining 26 half-cystine residues found by Amino acid analysis form intrachain bonds. Furthermore, the distribution of labeled residues among the individual chains indicates that each light chain is linked to a heavy chain by a single disulfide bond, and the two heavy chains are connected by two disulfide bridges.

When analyzing proteins with complex quaternary structure, it is advisable to separate them into their constituent subunits or Polypeptides. In the case of IMMUNOGLOBULINS, Selective reduction of interchain disulfide bonds was performed to dissociate the polypeptide chains [44, 46]. To obtain large, readily identifiable fragments suitable for subsequent cleavage into short cystine-containing peptides, native proteins are cleaved with Cyanogen bromide or Proteolytic Enzymes [19, 28, 42, 46, 47].

To assess how reliably the isolated cystine-containing peptides reflect The structure of the native protein, the peptide yield is of critical importance. Indeed, isolating a peptide in high yield provides the most compelling evidence that two half-cystine residues are linked in the native protein. A yield of 50% is generally considered quite satisfactory, though yields may be lower due to mechanical losses and incomplete cleavage reactions. A low peptide yield does not rule out alternative disulfide arrangements; therefore, the obtained data can only be considered conclusive in the absence of conflicting evidence—for instance, unless another cystine-containing peptide incorporating one of the identified half-cystine residues is found.