Cystine peptides separation

For the preliminary separation of a complex protein hydrolyzate into simpler peptide mixtures, ionophoretic methods are probably the most generally useful. Aromatic peptides may be separated by adsorption on charcoal and cystine peptides by oxidation. 

Consden and Gordon (1950) have studied the cysteic acid peptides derived from the cystine peptides of wool as described on p. 40. After a preliminary group separation of the cysteic acid peptides by iono-phoresis, each fraction was fractionated on paper chromatograms with phenol and collidine. In Table VII are listed the peptides considered to be probably present and their approximate yield. The results were more clear-cut in this case than with the aspartic and glutamic acid... 

Small molecules containing disulfide bonds (such as cystine-containing peptides) may be reduced and isolated simply by removing the immobilized reductant. Separation of reduced molecules from reductant is much more difficult if a soluble reducing agent is used with low-molecular-weight disulfides. 

Determination of iodo amino acids by HPLC with inductively coupled plasma (ICP)-MS detection had LOD 35-130 pg of I, which is about one order of magnitude lower than with UVD usually applied for these compounds175. Amino acids and peptides containing sulfur, such as cysteine, cystine, methionine and glutathione, can be determined after HPLC separation by pulsed electrochemical detection, using gold electrodes176. 

The A and B peptide chains in insulin are linked through disulfide bridges. Their presence was suspected from the change in molecular weight which followed the reduction of insulin. For quantitative analyses the S-S bridges had to be broken. Sanger, following the approach used by Toennies and Homiller (1942), oxidized the protein with performic acid, so that the half-cystines were converted to cysteic acid. After oxidation, insulin could be separated into its A and B chains, the A peptide with 20 amino acid residues and the B with 30. 

Peptide bonds are cleaved in a nonselective, but not in a completely random manner. Based on anchimeric side-chain assistance, steric factors, and bond strains, acid-labile peptide bonds are predicted to include sites containing Asp, Glu, Ser, Thr, Asn, Gin, Gly, and ProJ22l The disulfide topologies of circulin B and cyclopsychotride, backbone-cyclized peptides with three disulfide bonds, were determined by partial hydrolysis for 5 hours.[22 Occasionally, the bond between adjacent half-cystine residues is cleaved due to the nonselective nature of the mechanism of partial acid hydrolysis.[21] By this procedure, in all cases, a complex mixture of peptide fragments is produced which requires careful chromatographic separation by RP-HPLC for subsequent analysis by mass spectrometry (see Section 6.1.6.2.7). 

The primary level of structure in a protein is the linear sequence of amino acids as joined together by peptide bonds. This sequence is determined by the sequence of nucleotide bases in the gene encoding the protein (see Topic HI). Also included under primary structure is the location of any other covalent bonds. These are primarily disulfide bonds between cysteine residues that are adjacent in space but not in the linear amino acid sequence. These covalent cross-links between separate polypeptide chains or between different parts of the same chain are formed by the oxidation of the SH groups on cysteine residues that are juxtaposed in space (Fig. 4). The resulting disulfide is called a cystine residue. Disulfide bonds are often present in extracellular proteins, but are rarely found in intracellular proteins. Some proteins, such as collagen, have covalent cross-links formed between the side-chains of Lys residues (see Topic B5). 

Val-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn-COO-. When only the composition is known (i.e., the sequence is unknown), the amino acids are separated by commas and enclosed in parentheses, for example, (Ala,Cys2,Gly) means a peptide containing one Ala, two Cys, and one Gly in an unknown order. A "polypeptide chain" is, by convention, a continuous chain linked only by peptide bonds. A protein may have only one polypeptide chain and then the polypeptide and the protein are synonymous. In other cases, a protein may have more than one polypeptide chain, as does insulin. In such cases, the different peptide chains within a protein may be held together by noncovalent forces, as in the case of hemoglobin, sometimes supplemented by covalent cystine cross links, as in the case of insulin. In many cases, the noncovalent interactions allow more than one conformation and a protein may switch from one conformation to another as part of its function. 

These results indicate that insulin is built up of four open polypeptide chains, which seem to be held together by —S—S— bridges of cystine. These are present to the extent of six residues per molecule. By oxidation with performic acid (p. 27) it was possible to split these bridges and thus to liberate the separate peptide chains (Sanger, 1949a). The oxidized insulin was fractionated by precipitation methods and two main fractions were obtained, which appeared to represent the whole of the oxidized insulin. The properties of these two fractions, designated A (acidic) and B (basic), are summarized in Table VIII. The most probable composition of fraction A is,... 

---