Practical Protein Chemistry - A. Darbre 1989

Disulfide Bonds
Fractionation of Cystine-Containing Peptides
Detection of Cystine-Containing Peptides

During the Separation and purification of cystine-containing Peptides, a selective detection method is required. A widely used approach involves determining the Amino Acid Composition of fractions after peptide oxidation with performic acid [41]. Cystine-containing fractions are identified by the presence of cysteic acid residues in the peptide. It has been shown that sulfur in a partially oxidized state decolorizes an iodoplatinate solution [16]. This reaction can serve as a rapid and sensitive test for disulfides, although a similar reaction is given by sulfur in the form of thiols and thioethers. A sensitive fluorimetric detection method is based on fluorescence quenching upon the interaction of disulfides with fluorescein mercuriacetate in an alkaline medium [26]. Unfortunately, this Procedure has poor reproducibility.

4.5.2.1. Disulfide analysis [1]. This procedure is highly reproducible and can be automated. A peptide solution (including Chromatography fractions) is mixed with an equal volume of 6 M NaOH and incubated for ~30 min. The reaction is stopped by adding, with vigorous stirring, 0.3 ml of 2 M phosphoric acid containing 2×10^-3 M EDTA. To the resulting solution (pH 6.0–7.0), 0.1 ml of DTNB solution (1.0 mg/ml in 0.02 M sodium acetate, pH 5.5) is added, and the absorbance is measured at 412 nm. EDTA binds Metal Ions that catalyze thiol oxidation, thereby stabilizing the color. Nevertheless, after 60 min, the absorbance decreases by 1–2%. Glutathione (0–2.5×10^-2 mol/L) is used to construct the calibration curve.