Insulin hexameric

The van der Waals model of monomeric insulin (1) once again shows the wedge-shaped tertiary structure formed by the two chains together. In the second model (3, bottom), the side chains of polar amino acids are shown in blue, while apolar residues are yellow or pink. This model emphasizes the importance of the hydrophobic effect for protein folding (see p. 74). In insulin as well, most hydrophobic side chains are located on the inside of the molecule, while the hydrophilic residues are located on the surface. Apparently in contradiction to this rule, several apolar side chains (pink) are found on the surface. However, all of these residues are involved in hydrophobic interactions that stabilize the dimeric and hexameric forms of insulin. 

Ciszak, E., Beals, J. M., Frank, H. et al. (1995). Role of C-terminal B-chain residues in insulin assembly the structure of hexameric LysB28 ProB29-human insulin. Structure 3, 615. 

Insulin therefore consists of two peptide chains that are connected by two disulfide bonds, since the C-peptide is cleaved off. There are some species-specific differences in the amino acid sequence of the hormone. X-ray diffraction studies have shown that insulin occurs as a hexameric protein containing two Zn atoms. The dimers are first held by four hydrogen bonds and a hydrophobic bond along the sequence in the form of an antiparallel P sheet. The dimers then bind by interaction of the B -Ala, B -Leu, and B -Val residues. The core of the hexamer contains water. 

Native human insulin self-associates to hexameric unit, which limits its absorption rate from injection sites (e.g., subcutaneous or intramuscular space). Based on the... 

However, short-acting, regular soluble insulin is the only type that should be administered intravenously because the dilution causes the hexameric insulin to immediately dissociate into monomers. It is particularly useful for intravenous therapy in the management of diabetic ketoacidosis and when the insulin requirement is changing rapidly, such as after surgery or during acute infections. 

Liposomes were formed from 1,2-dipalmitoylphosphatidylcholine (DPPC) and cholesterol (Choi) and the effect of liposomal entrapment on pulmonary absorption of insulin was related to oligomerization of insulin (Liu et al. 1993). Instillation of both dimeric and hexameric insulin produced equivalent duration of hypoglycemic response. However, the initial response from the hexameric form was slightly slower than that from dimeric insulin, probably due to lower permeability across alveolar epithelium of the hexameric form caused by larger molecular size. The intratracheal administration of liposomal insulin enhanced pulmonary absorption and resulted in an absolute bioavailability of 30.3%. Nevertheless, a similar extent of absorption and hypoglycemic effects was obtained from a physical mixture of insulin and blank liposomes and from liposomal insulin. This suggests a specific interaction of the phospholipid with the surfactant layer or even with the alveolar membrane. 

Figure 7-18 Stereoscopic MolScript ribbon drawings of the B chains (A chains omitted) of (A) hexameric 2-zinc pig insulin. (B) A phenol complex of the same protein. Within each dimer the B chains are shaded differently. The Zn2+ ions are represented by white spheres and the coordinating histidine side chains are shown. Six noncovalently bound phenol molecules can be seen, as can several conformational differences. From Whittingham et al.B7 Courtesy of Peter C. E. Moody.

In addition to its function in catalysis, zinc often plays an important structural role, e.g., in the zinc finger transcriptional regulators (Fig. 5-38).k Zinc ions bind to insulin and stabilize its hexameric structure (Fig. 7-18)/ Six Zn2+ ions are present in the hexagonal tail plate of the T-even bacteriophage (Box 7-C) and appear to be essential for invasion of bacteria.131 In carnivores, the tapetum, the reflecting layer behind the retina of the eye of many animals, contains crystals of the Zn2+-cysteine complex. 

A, Inhibition of proteolytic enzymes B, dissociation of hexameric to monomeric form of insulin C, loosening of tight junctions D, increase in membrane fluidity due to cholesterol removal E, reversible ciliostasis F, mucoadhesion and prolonged residence time G, incorporation into lipid bilayer and membrane perturbation H, insulin internalization, increased transcellular transport and I, correlation with CMC. 

Since its discovery, isolation, and purification in the early twentieth century, insulin has been administered to diabetic patients exclusively by injection until the recent introduction of inhaled insulin. Insulin possesses certain physiochemical properties that contribute to its limited absorption from the gastrointestinal tract, and requires subcutaneous injection to achieve clinically relevant bioavailability. With a molecular size of 5.7 kDa, insulin is a moderately sized polypeptide composed of two distinct peptide chains designated the A chain (21 amino acid residues) and the B chain (30 amino acid residues) and joined by two disulfide bonds. Like all polypeptides, insulin is a charged molecule that cannot easily penetrate the phospholipid membrane of the epithelial cells that line the nasal cavity. Furthermore, insulin monomers self-associate into hexameric units with a molecular mass greater than 30 kDa, which can further limit its passive absorption. Despite these constraints, successful delivery of insulin via the nasal route has been reported in humans and animals when an absorption enhancer was added to the formulation. 

To explain the complex of phenomena observed in Fig. 2.5, we postulate that insulin is present in the subcutaneous depot in three different forms free insulin on dimeric form, free insulin on hexameric form, and insulin bound to various substances and surfaces in the tissue. Moreover, we assume that only dimeric insulin can diffuse across the capillary wall. In qualitative terms, our explanation is then ... 

At high insulin concentrations most of the insulin is free, and the chemical balance between the two-multimeric forms is shifted towards hexameric insulin. Since hexameric insulin is assumed to not be absorbed, the fractional absorption rate is small. However, as the insulin concentration in the depot gradually falls, the chemical balance is shifted towards the dimeric form, and the absorption rate increases. 

The reduced absorption rate observed in the initial phases at high insulin concentrations is the dimeric absorption rate multiplied by the fraction of insulin on dimeric form. This allows us to determine the equilibrium constant Q 0.06 (ml/IU)2 for the mass balance between dimeric and hexameric insulin in the tissue. 

Here, Ch, Cb, and CB denote the local concentrations of hexameric, dimeric, and bound insulin, respectively, and C is the binding capacity for insulin in the tissue. Only dimeric and hexameric insulin is supposed to diffuse in the tissue. The corresponding diffusion terms are DdV2Cd and DhV2C, with DB and Dh being the two diffusion constants and V2 = the Laplacian operator. For the... 

To complete this discussion, let us finally note that our model (or the underlying hypotheses) allows us to predict the absorption curves for insulin concentrations outside the range of our experimental curves (Fig. 2.5). For a bolus injection of 0.1 ml of 100 IU/ml soluble insulin, for instance, we expect the initial fraction of hexameric insulin to be even higher than for 40 IU/ml. Hence, the fractional absorption rate is very low at the beginning, and it takes an even longer time before the insulin concentration reduces to such a level that dimeric insulin dominates,... 

Use of Near UV CD Spectra to Probe Aggregation in Polypeptides. Near UV CD spectroscopy can be employed to monitor the aggregation of a polypeptide species. Upon association, the aromatic groups will be in different environments than in the isolated monomers and the CD may change dramatically, especially in small peptides. This can be seen for insulin, where the near UV CD intensity of a monomeric form is only [e] - -150 deg cm2 dmoH. Yet, the hexameric form displays a signal nearly twice as intense [110-112],... 

Peptide and protein instability in vitro is manifested by the tendency of such molecules to undergo selfassociation in solution, resulting in the formation of multimers and, in the extreme, aggregation and precipitation. For example, insulin at pH 7 exists predominantly as hexameric aggregates, which are too large to be absorbed. 

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. ... 

Figure 9.19 offers a diagrammatic representation of the events following s.c. administration of a soluble human insulin existing initially as a hexameric zinc-insulin complex. 

Propylene glycol, glycerol, nonionic and ionic surfactants and calcium ions have been used in formulations to achieve greater stability, reducing fibril formation, but the most successful strategy is the addition of calcium ions or zinc, which appear to protect the hexameric form of the insulin (see section 9.4.4). 

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. 

Sulfatide binds directly to insulin, and sulfatide, but not its galactosyl-ceramide precursor, was able to compete with monoclonal antibodies directed against a subdomain of the insulin molecule (Osterbye et al., 2001). Sulfatide specifically promotes in vitro refolding of proinsulin into its zinc-dependent hexameric form while without a sulfatide or in the presence of galactosylcer-amide, proinsulin dimers and hexamers were nearly absent (Osterbye et al.,... 

An additional form of insulin, partiaiiy unfoided insuiin, can form a viscous or insoiubie precipitates known as fibriis. Shielding of hydrophobic domains is the principal driving force for the aggregation. Further studies reveaied that when the exposed hydrophobic domain (A2, A3, B11, and B15) interacts with the normally buried aiiphatic residues (A13, B6, B14, and B18) in the hexameric structure, fibrils form (Fig. 32.1) (39). Fibrils also have been studied by electron microscopy, and packing considerations in the crystal lattice explain why fibril formation is accelerated when insulin is in the monomeric state (40). Insulin fibrils do not resuspend on shaking thus, they are pharmaceutically inactive. 

Near-UV CD has also been useful in monitoring conformational transitions among insulin conformers in solution. Three hexameric conformers, Tfi, T3R3, and R, have been observed in crystals. Extensive studies by Wollmer and co-workers " have shown that the T R transition is marked by an increase in magnitude of the negative CD between 250 and 255 nm. This transition can be induced by binding of polarizable anions, with the effectiveness following the Hofmeister series, and by phenol derivatives. The transition in the crystal involves conversion of residues... 

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