Insulin hexamers

Proinsulin is proteolytically processed in the coated secretory granules, yielding mature insulin and a 34-amino acid connecting peptide (C peptide, Figure 11.1). The C peptide is further proteolytically modified by removal of a dipeptide from each of its ends. The secretory granules thus contain low levels of proinsulin, C peptide and proteases, in addition to insulin itself. The insulin is stored in the form of a characteristic zinc-insulin hexamer, consisting of six molecules of insulin stabilized by two zinc atoms. 

The concentration of insulin present in soluble insulin preparations (i.e. fast-acting insulins), is much higher (approximately 1 x KT2 3 mol I ). At this concentration, the soluble insulin exists as a mixture of monomer, dimer, tetramer and zinc-insulin hexamer. These insulin complexes have to dissociate in order to be absorbed from the injection site into the blood, which slows down the onset of hormone action. 

Fig. 61. (a) The insulin hexamer (b) the insulin monomer. From Blundell et al. (1972), with permission. 

Although insulin was first crystallized in 1926, the factors promoting crystal growth were poorly understood and yielded inconsistent results. It was almost 10 years later when researchers discovered that the addition of zinc to a crude extract promoted reproducible crystallization (zinc addition yields a characteristic rhombohedral crystal, the basic crystal unit being the insulin hexamer, stabilized by the two zinc atoms). 

The crystal structure of this protein has been shown to depend on the salt solution from which it is crystallized.45 When crystallized from (NH4)2S04, the insulin hexamer is held together in part by two Zn2+ ions. These can be visualized as being at either end of a cylinder, and each Zn2 has as a ligand one histidine imidazole nitrogen atom from one of three chains. Thus a histidine (his B.10) of each of the six chains is coordinated to Zn2+, three at each Zn2+. The Zn2+ ions occupy octahedral (trigonally distorted) sites overall. 

Soybean-derived sterol mixture (SS), soybean-derived steryl glucosides (SG), and their individual components have been extensively studied for their ability to promote the nasal absorption of drugs, particularly insulin [79,80], Maitani et al. [79] demonstrated that the nasal administration of SG plus insulin to rabbits resulted in significant reductions in blood glucose. The effect of SG was dose dependent to 1%, with a plateau being reached thereafter. Muramatsu et al. [81] have demonstrated that SG perturbs the phospholipids in artificial membranes (i.e., liposomes). Furthermore, circular dichroism studies with insulin in the presence or absence of SG have indicated that the enhancer had little effect on the dissociation of insulin hexamers to monomers. These results suggest that the action of SS and SG involves interaction with the nasal membrane rather than interaction with insulin molecules. 

FIGURE 8.21. The use of a rotation function. Shown in (a) is the diffraction pattern of the 2Zn insulin hexamer (Ref. 86). Monomers form dimers by way of a noncrystal-lographic twofold axis. Dimers form hexamers by way of a crystallographic threefold axis perpendicular to the twofold axis. The twofold axes (indicated by spikes in the diffraction pattern) are highlighted in (a) through (d) by arrows, (b) The corresponding Patterson function [calculated from the diffraction data in (a)]. Barker planes between equivalent atoms in the two molecules are indicated by----------. 

Z. Shao, Y. Li, R. Krishnamoorthy, etal. Differential effects of anionic, cationic, nonionic, and physiologic surfactants on the dissociation, alpha-chymotryptic degradation, and enteral absorption of insulin hexamers. Pharm. Res., 10, 243-51 (1993)... 

Solution structures of the R-6 human insulin hexamer. Biochemistry, 36, 9409-9422. 

Figure 3 The structural levels of proteins, exemplified by human insulin in the T6 form. (A) Primary structure residues 15-18 of human insulin B-chain, shown as sticks. (B) Secondary structure residues 8-20 of the B-chain form an a-helix, here depicted as a superposition of sticks, and a cartoon-representation. (C) Tertiary structure insulin A- and B-chains fold up to a monomer, which is assumed to be the active form, binding to the insulin receptor. Insulin can exist in different oligomeric forms, depending on formulation and protein concentration. (D) The Zn -stabilized hexamer form is shown. 2 Zn ions are bound per insulin hexamer (only one Zn2" "-ion is visible in this view). The hexamer is a trimer of dimmers. Figure based on pdb-file IMSO, produced in Pymol. Source Bente Vestergaard, Biostructural Research, Faculty of Pharmaceutical Sciences, University of Copenhagen.

In addition to the above-mentioned excipients and functions, preservation of formulations in multiple dose containers is also required. Therefore, antimicrobial preservatives such as phenol, methyl-, and propylparabens are added. Apart from their formulation function the excipients also interact specifically with the protein and can thereby cause alterations in its function and stability (7). For example, phenol has an influence on the stability and conformation of the insulin hexamer. The addition of phenol shifts the structure from the less stable T3 to the more stable R6 (71). 

Fig. 9. Time-resolved RSSF spectra (A), difference spectra (B, C) and single-wavelength time courses (D) for the reaction of l-(2-pyridylazo)-2-naphthol (PAN) with the Zn(II)-T6 insulin hexamer (59). The expanded difference spectra shown in (C) and the singlewavelength time course measured at 500 nm in (D) establish that reaction occurs via the formation and decay of an intermediate. Concentrations after mixing in (A-C) [Zn(II)-T6] = 2.5 iM, [PAN] = 15 iM. In (D), [Zn(II)-T6] = 2.5 iM, [PAN] = 15 pM. A 50 mM pH 8.0 Tris-HCl buffer at 25°C was used in both experiments. Unpublished work of G. G. Gould and M. F. Dunn.

Insulin exists as a hexamer, both in solution and as a crystal. Bergeron et al. report the solubility of insulin in aqueous solution to be about 0.122 mg/mL at lO C and 0.182 mg/mL at 25 C, and the molecular weight of the insulin hexamer to be 34 800. Use these data to estimate the heat of fusion of the insulin hexamer. 

Since the molecular weight of the insulin hexamer is so high, its mole fraction at both temperatures is very small. For example, at the higher concennation of 0.182 mg/mL,... 

Insulin is a peptide hormone (MW = 5734) it contains an A peptide of 21 amino acids and a B subunit of 30 amino acids, linked by one intrasubunit and two intersubunit disulfide bonds (Figure 60-1). The two chains of insulin form a highly ordered structure with a-helical regions in each of the chains. In solution, insulin can exist as a monomer, dimer, or hexamer. Two molecules ofZn are coordinated in the hexamer, and this form of insulin presumably is stored in the /3 cell granules. Insulin hexamers also comprise most of the highly concentrated preparations used for therapy. As the concentration falls to physiological levels (nM), the hormone dissociates into monomers, which likely are the biologically active form. 

Hypoglycemic Agent. Monomeric form of regular insulin hexamer. Absorbed more rapidly than regular insulin. Lsy28 and Pro29 swapped in B chain... 

The importance of zinc ions for stabilizing insulin preparations has been known since the first reported crystallization of insulin in the presenoe of zino ions in 1934 (36). Suspensions of zinc insulin were used at that time. Presently, all pharmaceutical preparations are either solutions of zino insulin or suspensions of insoluble forms of zino insulin. A longer-acting and more stable form of insulin is protamine zinc insulin, which is prepared by precipitating insulin in the presence of zinc ions and protamine, a basic protein. This precipitate is known to contain two zinc ions per insulin hexamer. A somewhat shorter-acting and more useful preparation is neutral protamine Hagedorn (NPH) insulin, which includes m-cresol... 

Smith GD, Swenson DC, Dodson EJ, et al. Structural stability in 4-zinc human insulin hexamer. Proc Natl Acad Sci USA 1984 81 7093-7097. 

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