Insulin dimer

Another degradation reaction observed in suspension was the formation of covalent insulin dimers [134][136], These involve isopeptide links between two insulin molecules, that result from a transamidation reaction mainly between the B-chain N-terminus of one insulin molecule, and one of the four amide side chains in the A-chain (principally AsnA21) of the second insulin molecule. 

Pocker, Y Biswas, S.B. Selfassociation of insulin and the role of hydrophobic bonding a thermodynamic model of insulin dimerization. Biochemistry 1981, 20, 4354-4361. 

Chromatographic or electrophoretic analysis of conventional insulins generally yields three major fractions or bands a, b and c). Fraction a contains high molecular mass material which can be removed from the product by additional recrystallization steps. The major components of fraction b are proinsulin and insulin dimers, while insulin, as well as slightly modified forms of insulin (e.g. arginine-insulin and desamido-insulin), are found in fraction c. 

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.

Bertera S, Geng X, Tawdrous Z, Bottino R, Balamurugan AN, Rudert WA, Drain P, Watkins SC, Trucco M. Body window-enabled in vivo multicolor imaging of transplanted mouse islets expressing an insulin-dimer fusion protein. Biotechniques 2003, 35, 718-722. 

Data of Tanford and Epstein (1954 see also Frederioq 1954, 1956), calculated for an insulin dimer of molecular weight 11,466. The zinc insulin preparation contained one zinc atom per dimer molecule. The pKint values are for the zinc-free protein. 

Baker et al. (1987, 1988) described a 1.5 A resoludon structure of 2Zn-insulin. They located 282 of the estimated 285 waters per insulin dimer in the crystal. These were distributed among 349 sites 217 of occupancy 1.0 126 of occupancy 0.5 five of occupancy 0.33 and one of occupancy 0.25. There was evidence for ordered water at a distance 8 A from the protein surface. Nearly 100 waters were bonded only to other waters. The extent of order of the water, judged by B values, increased with an increased number of interactions with the protein. The waters bonded to the protein act as anchors for chains of less well-ordered waters, which are often linked by threads of density, possibly representing paths along which the less-ordered waters are found. There were alternate water positions, sometimes collected into networks of partially occupied sites. Cyclic water structures were found. The protein—water contacts showed preferred geometries. Baker et al. (1988) gave particularly elegant descriptions of the crystal water. 

Aggregation of a protein may also be desired when the aggregate is a more stable form of the protein. For example, insulin is formulated in the presence of Zn " ", which coordinates insulin dimers to form an ordered hexameric form of the protein. Zn added to the formulation has been shown to increase the physical stability of an insulin solution. ... 

Representations in a stylised diagrammatic form of three proteins (interleukin-l/J, zinc insulin dimers, and the Fc fragment of immunoglobulin) are shown in Fig. 11.3. The nature of the three-dimensional stmcture shows how difficult it is to define proteins in conventional ways, and how they must be considered in a new light as pharmaceutical... 

Figure 11.3 Ribbon diagrams of (a) interleukin 1-/ , (b) zinc-insulin dimer and (c) the Fc fragment of immunoglobulin.

Figure 2.11 In situ STM of human insulin on single-crystal Au electrode surfaces [1 70], At left is a structural representation of the expected dominating insulin dimer form (PDB 1 B9E). The A- and B-chains and the three disulfide groups in each monomer (blue, red,...

Insulin monomers undergo noneovalent dimerization by formation of antiparallel /1-pleated sheet associations between monomers involving the C-terminal portion of the B chain. As discussed earlier, lispro insulin, in which the B28 and B29 is reversed from the normal prolyl and lysyl sequence, does not dimerize. Three insulin dimers subsequently self-associate to form hexamers in the presence of Zn +. The Zn + hexameric array of insulin probably gives the /3-cell granule its unusual morphologic characteristics. 

Roth, R. A., Cassell, D. J., Morgan, D. O., Tatnell, M. A., Jones, R. H., Schtittler, A., Brandenburg, D. Effects of covalently linked insulin dimers on receptor kinase activity and receptor down regulation. FEES Lett. 1984,170, 360-364. 

Only the insulin monomer is able to interaot with insulin receptors, and native insulin exists as a monomer at low, physiologioal oonoentrations (<0.1 pM). Insulin dimerizes at the higher oonoentrations (0.6 mM) found in pharmaceutioal preparations, and at neutral pH in the presence of zinc ions, hexamers form (34). These zino-associated hexamers also are the storage form of insulin in p cells. At concentrations greater than 0.2 mM, hexamers form even in the absence of zino ions. 

The A chains in each molecule of the 4Zn insulin dimer are quite similar to each other in conformation, although not in disposition to the B chains. In molecule I the A chain has moved away from the B chain (Fig. 3) as a result of the movement of the A7-B7 disulfide link, which causes a cleft to appear in this molecule. This variation in structure of molecule I, particularly the formation of the cleft, does not seem to impair biological activity of 4Zn insulin preparations there is good evidence that on dissociation of the 4Zn hexamers the monomeric conformations are closely similar to that of molecule II in the two hexameric forms. Indeed, it has been suggested that the flexible character of insulin could be an important factor in generating biological response. 

X-ray, circular dichroism, and centrifuge studies have demonstrated that most mammalian and fish insulins form zinc insulin hexamers similar to those of porcine insulin (Blundell et al, 1972 Blundell and Wood, 1975), with the exception of hagfish insulin, which produces only dimers (Cutfield et al, 1974 Peterson et al, 1974), and guinea pig (Zimmerman et al, 1972), casiragua (Horuk et al, 1979), and porcupine (Horuk et al, 1980) insulins, which exist only as monomers. A complete structural analysis of hagfish insulin dimers shows that, unlike porcine insulin, the two molecules of the dimer in the crystals are exactly equivalent and resemble molecule II of the asymmetric dimer of porcine insulin. This is similar to the structure of porcine insulin in solution as indicated by circular dichroism studies (Wood et al, 1975 Strickland and Mercola, 1976). 

As a result of derivatization of insulin during isolation and purification steps, and during storage of the pharmaceutical preparations, therapeutic insulin contains smaller amounts of desamido insulins, covalent insulin dimers, and other insulin derivatives. Physical and chemical stability of insulin in formulations for injection therapy has recently been reviewed in a previous volume of this series (Brange and Langkjser, 1993) and in a monograph (Brange, 1994). 

Although covalent oligomers such as dimers may be relatively small and may remain fully soluble, they can possess enhanced toxicity and immunogenicity relative to the monomer. It is not trivial to isolate and characterize individual protein oligomers to the extent of resolving toxicology in preclinical models, so published examples are not plentiful. However, the rich history of insulin as a therapeutic protein provides a well-characterized example. Covalent insulin dimers have been detected in the blood of insulin... 

Histamine gives a stable complex with Co +, as it gives the same sort of complex with Cu + (see above). It is interesting to link this result with the fact that insulin dimerizes through the presence in its structure of histidine and through the participation of the zinc ion Zn +, which is divalent. 

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