Protein sequencing disulfide bond cleavage

The first step, protein purification, is discussed in chapter 5. Once the protein is pure, sequence analysis can begin, with cleavage of the disulfide bonds. Cleavage is achieved by oxidizing the disulfide linkages with performic acid (fig. 3.17). Sometimes this step results in the production of two or more polypeptide chains, in which case the individual chains must be separated. 

Once the protein s primary sequence has been determined, the location of disulfide bonds in the intact protein can be established by repeating a specific enzymatic cleavage on another sample of the same protein in which the disulfide bonds have not previously been cleaved. Separation of the resulting peptides shows the appearance of one new peptide and the disappearance of two other peptides, when compared with the enzymatic digestion product of the material whose disulfide bonds have first been chemically cleaved. In fact, these difference techniques are generally useful in the detection of sites of mutations in protein mole-... 

In order to assign the disulfide bonds of these molecules fast atom bombardment mass spectrometry (FABMS) which has been used not only to confirm amino acid sequence data but also to elucidate post-translational modifications of proteins, such as disulfide bonds, has been employed. For this purpose a sample of native Er-2, containing four methionines, was subjected to CNBr cleavage and without further fractionation directly... 

After the sequence of the individual chains has been determined, a sample of the protein is partially hydrolyzed without cleavage of the disulfide bonds, and the fragments containing the intact disulfide bonds are sequenced. 

Table I summarizes the sequencing results from alcohol dehydrogenase and the N-terminally blocked glycoprotein ovalbumin. The probable identity of these fragments is indicated. All fragments identified for both proteins by N-terminal sequencing corresponded to cleavage after cystine. The data from ovalbumin are particularly interesting. The structure of ovalbumin is well characterized (5) and contains only one disulfide bond between Cys 73 and Cys 120 yet sequence was obtained following Cys 11 and Cys 30. The bands for these fragments appeared more slowly than the odiers and were fainter in appearance.

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