Insulin disulfide bridges

Disulfides. As shown in Figure 4, the and h-chains of insulin are connected by two disulfide bridges and there is an intrachain cycHc disulfide link on the -chain (see Insulin and other antidiabetic drugs). Vasopressin [9034-50-8] and oxytocin [50-56-6] also contain disulfide links (48). Oxidation of thiols to disulfides and reduction of the latter back to thiols are quite common and important in biological systems, eg, cysteine to cystine or reduced Hpoic acid to oxidized Hpoic acid. Many enzymes depend on free SH groups for activation—deactivation reactions. The oxidation—reduction of glutathione (Glu-Cys-Gly) depends on the sulfhydryl group from cysteine. 

FIGURE 15.3 Proinsulin is an 86-residue precursor to insulin (the sequence shown here is human proinsulin). Proteolytic removal of residues 31 to 65 yields insulin. Residues 1 through 30 (the B chain) remain linked to residues 66 through 87 (the A chain) by a pair of interchain disulfide bridges. 

These steps can be repeated to add one amino acid at a time to the growing chain or to link two peptide chains together. Many remarkable achievements in peptide synthesis have been reported, including a complete synthesis of human insulin. Insulin is composed of two chains totaling 51 amino acids linked by two disulfide bridges. Its structure was determined by Frederick Sanger, who received the 1958 Nobel Prize in chemistry for his work. 

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. 

The kinetics of disappearance from the circulation of intravenously administered human insulin (Fig. 6.32) is nonlinear [145]. Within a few minutes after injection, it becomes localized in the liver, heart, and kidneys, where it is rapidly metabolized. Indeed, the hepatic extraction could be as high as 70% on a single passage, whereas kidneys could account for 10-40% degradation. Enzymatic reduction of the disulfide bridges appears to be the first step in the in vivo metabolism of insulin, although this reaction appears of limited significance under in vitro conditions. 

F ig U re 10.2 The primary structure of bovine insulin. This molecule possesses two polypeptide chains, labeled A and B. These are joined by two disulfide bridges between Cys amino acid residues. There is a third disulfide bridge linking two Cys residues in the A chain. 

Human insulin is composed of two straight-chain polypeptides joined by disulfide bridges. 

Porcine insulin and bovine insulin for drug use are extracted from the pancreas of pigs and cattle respectively. More frequently, human insulin is now employed. This is produced by the use of recombinant DNA technology to obtain the two polypeptide chains, and then linking these chemically to form the disulfide bridges... 

Insulin, a pancreatic hormone, is a specific antidiabetic agent, especially for type I diabetes. Human insulin is a double-chain protein with molecular mass around 6000 that contains 51 amino acids (chain A—21 amino acids, chain B—30 amino acids), which are bound together by disulfide bridges. 

Scheme 24 Synthesis of Human Insulin by Regioselective Disulfide Bridging of the A- and B-Chains1 1 31...

Cleavage of the Trt group of one chain 54 with a weak acid to give 55 and its subsequent thiolysis of the. S -SPy derivative of the second chain 57 directs the formation of the first interchain disulfide bond in 58. The second interchain disulfide bridge is formed between the two Acm-protected cysteine residues of the [bis(Acm), bis(tBu), mono-disulfide]-hetero-dimer 58 by treatment with iodine. Finally, treatment of 59 with chlorosilane/sulfoxide produces the third disulfide bond between the two tBu-protected cysteine residues yielding human insulin (42). 

The bis-disulfide bridged human insulin 59 (l.Omg, 0.17pmol) in TFA (0.6mL) was treated with Me-SiCl3 (5 pL 250 equiv) in the presence of PhS(0)Ph (0.7 mg, 20 equiv) at 25 °C for 15 min. NH4F (3 mg) was added to the mixture, and the solvent was removed in under reduced pressure. The residue was dissolved in 50% AcOH (1 mL) and the soln was gel-filtered on Sephadex G-25. The product 42 was further purified by semipreparative HPLC yield 0.6mg (61%) the synthetic human insulin was characterized by amino acid analysis and FAB-MS, it exhibited identical chromatographic and biological properties as a reference compound. 

Biopharmaceuticals based on natural proteins and peptides are often called by the same name as the biologic natural material despite differences in one or more amino-acid residues. For example, insulin, which regulates blood glucose and is used clinically to treat type 1 diabetes and some cases of type 2 diabetes, has several variants that are approved for human use. Insulin contains two polypeptides, A and B chains (Figure 1.2), that are linked together by two disulfide bridges to assume a biologically active conformation. Compared with human insulin, insulin extracted from beef tissue exhibits threonine alanine and isoleucine valine substitutions at posi-... 

Insulin is composed of 51 amino acids arranged in two polypeptide chains, designated A and B, which are linked together by two di- sulfide bridges (Figure 23.3A). The insulin molecule also contains an j intramolecular disulfide bridge between amino acid residues of the A chain. Beef insulin differs from human insulin at three amino add positions, whereas pork insulin varies at only one position. 

Figure 7-17 The structure of insulin. (A) The amino acid sequence of the A and B chains linked by disulfide bridges. (B) Sketch showing the backbone structure of the insulin molecule as revealed by X-ray analysis. The A and B chains have been labeled. Positions and orientations of aromatic side chains are also shown. (C) View of the paired N-terminal ends of the B chains in the insulin dimer. View is approximately down the pseudo-twofold axis toward the center of the hexamer. (D) Schematic drawing showing packing of six insulin molecules in the zinc-stabilized hexamer.

Insulin, RMM about 6000, is made up of two chains of amino adds joined by disulfide linkages. The sequence of amino acids in the two chains (termed A for acidic and B for basic) and the arrangement of the three disulfide bridges were worked out by Sanger and associates in the period 1945-1955.1229 The complete synthesis of both ovine and human insulin was achieved in 1963.1230,1231... 

Figure 25-8 Amino-acid sequence in beef insulin. The A chain of 21 amino-acid residues is linked to the B chain of 30 residues by way of two disulfide bridges.

The disulfide bridges in some proteins are between different peptide chains. Insulin, for instance, has two interchain as well as one intrachain S—S bridges (Figure 25-8). 

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