Cleavage of disulphide bonds

As indicated in Section 5.2, disulphide bonds may be either intra- or inter-chain ones and end-group analysis might permit differentiation between these possibil- 

The 3-phenyl-2-thiohydantoins can be separated and identified by TLC on silica gel. If the latter contains a fluorophore, the thiohydantoins quench the fluorescence 

Obviously, incomplete reaction and losses during manipulation prevent the yield of 3-phenyl-2-thiohydantoin from reaching 100%. With each cycle of the Edman method, the yield of product derived from the newly exposed TV-terminus will decrease. In addition, small amounts of the 3-phenyl-2-thiohydantoins corresponding to earlier positions in the sequence will be formed as a consequence of incomplete reaction at each cycle. If the fraction of peptide that reacts with phenyl isothiocyanate and gives the relevant 3-phenyl-2-thiohydantoin is x, then the yield at any stage is 

Three main methods of coupling are used to attach peptides covalently to those 

After polypeptides have been separated by polyacrylamide electrophoresis, they can be electrophoretically transferred to a glass-fibre filter paper that has been treated with 3-aminopropyltriethoxysilane and covalently bonded using 4,4 -phenylene diisothiocyanate. The glass-fibre disc loaded with as little as 10 ag of polypeptide can be placed directly into a gas-phase sequenator (Aebersold et al., 1988). 

---

It is usual to effect cleavage of disulphide bonds by reduction or oxidation. Addition of a large excess of a thiol such as 2-mercaptoethanol or 1,4-dithiothreitol to a polypeptide reduces cystine residues to cysteine (Scheme 5.1). In order to prevent reoxidation in air, the generated thiol groups are blocked, usually by reaction with iodoacetic acid. The product yields S -carboxymethylcysteine (5.9) on hydrolysis for amino-acid analysis. Alternatively, oxidative cleavage of disulphide bonds can be achieved with performic acid each half of the cysteine residue is converted into a residue of cysteic acid (5.10). 

---