Practical Protein Chemistry - A. Darbre 1989

Disulfide Bonds
Determination of the Number of Disulfide Bonds

The number of Disulfide Bonds is determined by measuring the content of carboxymethylcysteine residues in a protein, i.e., by analyzing the Amino Acid Composition of the protein before and after its complete Reduction and Carboxymethylation. In the case of multi-subunit Proteins, to gain further insight into the distribution of disulfide bonds, they are fractionated into their constituent polypeptide chains. Half-cystine residues are quantified by The amount of label incorporated following reduction and subsequent alkylation with radiolabeled SH-Reagents. Reliable results are achieved using [¹⁴C]iodoacetamide (1.2 μCi/μmol) and [¹⁴C]iodoacetic acid (0.5 μCi/μmol) [19, 31].

The content of disulfide groups in a protein can be determined spectrophotometrically or fluorimetrically. The most widely used approach is based on the spectrophotometric quantification of disulfide groups using Ellman's reagent [14]. In [29], this technique was employed to determine the number of intra- and interchain bonds in human immunoglobulin M (IgM). The reduced (or native) protein was precipitated on ice with 5% trichloroacetic acid (TCA); the precipitate was collected by centrifugation and washed five times with a TCA solution to remove the reducing agent. The pellet was then dissolved in a small volume of 5 M guanidine, the solution was clarified by centrifugation, and the protein concentration was determined from absorbance at 280 nm. An equal volume of 0.1 M Tris-HCl (pH 9.1) containing 5 M guanidine and 2X10^-4 M 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was added to an aliquot of the protein solution, adjusting the pH of the reaction mixture to 8.1. The content of sulfhydryl groups was calculated from the absorbance at 412 nm using a molar extinction coefficient for 2-nitro-5-benzoate of ε = 13,600. Disulfide groups can also be determined by the quenching of fluorescein mercuriacetate fluorescence [26].