Insulin dimers, covalent

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. 

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... 

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. 

Precipitation is a macroscopic process characterized by a visible change of an otherwise clear solution in the form of an increase in solution viscosity, clouding of solution, or phase separation of solid materials. The formation of precipitates may be due to covalent polymerization and/or noncovalent aggregation (Section 4.3.2.2). In covalent polymerization, dimer formation usually precedes the precipitation, and the phenomenon is best illustrated by insulin, which has been extensively reported to form covalent dimers. 

Insulin is composed of two peptide chains covalently linked by disulfide bonds (Figures 5.17 and 6.35). This monomer of insulin is the active form that binds to receptors in target cells. However, in solution, insulin spontaneously forms dimers, which themselves aggregate to form hexamers. The surface of the insulin molecule that self-associates to form hexamers is also the surface that binds to insulin receptors in target cells. Thus, hexamers of insulin are inactive. 

Weiland M, Brandenburg C, Brandenburg D et al (1990) Antagonistic effects of a covalently dimerized insulin derivative on insulin receptors in 3T3-L1 adipocytes. Proc Natl Acad Sci USA 87 1154-1158... 

Quaternary structure. Due to non-covalent interactions, many proteins assemble to form symmetrical complexes (oligomers). The individual components of oligomeric proteins (usually 2-12) are termed subunits or monomers. Insulin also forms quaternary structures. In the blood, it is partly present as a dimer. In addition, there are also hexamers stabilized by Zn ions (light blue) (3), which represent the form in which insulin is stored in the pancreas (see p.l60). 

Strickley, R.G., and B.D. Anderson, Solid-state stability of human insulin. II. Effect of water on reactive intermediate partitioning in lyophiles from pH 2-5 solutions stabilization against covalent dimer formation. J Pharm Sci, 1997.86(6) 645-53. 

Finally, some mammalian receptors (certain NGF and interleukin receptors) are apparently capable of signalling with only one chain. However, in these cases, signalling seems to require the formation of heterodimers with accessory proteins, e.g. co-receptors, or interactions wdth other molecules, such as proteoglycans. Furthermore, there are the insulin receptors, which are constitutive dimers with the monomers covalently linked by... 

Intrinsic tyrosine kinases are covalently incorporated in the intracellular domains of some receptors, such as epidermal growdh factor receptor and the insulin receptor. When such receptor tyrosine kinases dimerize, cross-phosphorylation occurs. The phosphorylated tyrosines in activated receptor tyrosine kinases serve as docking sites for SH2 domains present in numerous signaling proteins and permit further propagation of the signal. A prominent component of such pathways is the small GTPase Ras. The Ras protein, like the G subunit, cycles between an inactive form bound to GDP... 

R. T. Darrington and B. D. Anderson, Evidence for a common intermediate in insulin deamidation and covalent dimer formation Effects of pH and aniline trapping in dilute acidic solutions, J. Pharm. Sci. 84, 275-282 (1995). 

Besides insulin, other biopharmaceuticals known to exhibit covalent polymerization include recombinant tumor necrosis factor-alpha, which forms nonreducible dimers and oligomers [98], and human insulin-like growth factor I, which is prone to covalent aggregation [99]. 

Porcine insulin Deamidation, fragmentation, and covalent dimerization during release (28)... 

The globular proteins sometimes consist of an assembly of a small number of identical or very similar sub-units, not linked together by covalent bonds. This is usually designated as the quaternary structure. Hemoglobin consists of 4 units of the type indicated in Figure 10.18b. Insulin readily forms a dimer from the two units in Figure 10.19. 

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