Monomers, insulin

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

The insulin monomer is of 6000 molecular weight, and either the monomer or the dimer is the active hormone. Thus in fact the zinc that binds the molecule into hexamers is not of physiological interest. What is... 

Insulin monomers are in equilibrium with more stable dimers and hexamers with known association constants fCdimer= 1.1 x 105 m-1, fChexamer = 2.89 x 108 m-2. 

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. 

Fig. 11.10 Insulin monomer structure with the three amino acids involved in polymer conjugation. A1 Gly is located in a hindered hydrophobic pocket, the e-amino group of B29 Lys is the most exposed and available to conjugation.

Bodan, W., Sitkowski, J., Bednarek, E., Tamowska, A., Kawecki, R., and Kozerski, L (2008) Structure of human insulin monomer in water/acetonitrile solution. Journal of Biomolecular NMR, 40, 55-64. 

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. 

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. 

Figure 1. Insulin monomers (a) associated through hydrophobic interactions and as antiparallel i -pleated sheet to dimers (b) and, in the presence of zinc, to 2Zn insulin hexamers (c). View along the crystallographic threefold axis. From Blundell et al. (1972).

Figure 2. The crystal structure of the insulin monomer viewed perpendicular to the threefold axis. The backbone of the A chain is represented by a double line and that of the B chain by a heavy line. From Blundell et al (1972).

More recently, manipulation of the structure of the B-chain, aiming at decreasing the potential for P-sheet interactions of the peptide backbone between insulin monomers, has also resulted in insulins with decreased tendency to self-associate. Examples of such analogs are the LysPro insulin (Brems et al, 1992), in which the B28 and B29 residues are interchanged, as is the case in insulin-like growth factor-I, and analogs characterized by deletion of one of the amino acid residues in the B25 B28 sequence (Balschmidt and Brange, 1992). 

Fig. 105. Relation between. time and the number, n, of exchanged hydrogen atoms per molecule of insulin monomer or of oxidized A chain (Linderstr0m-Lang, 1955a).

Rational protein engineering has been successfully used in a variety of problems of interest in biomedical engineering. One of the first, and perhaps the most widely used examples can be found with the protein engineering of human insulin for the treatment of diabetes [ 3 ]. Native insulin has evolved to form dimers and hexamers, so that it can be produced and stockpiled in the pancreas before it is needed for release in the body. When purified insulin is injected subcutaneously following a meal as a treatment for diabetes, only the active monomer form is desired, and thus the formation of dimers and hexamers can slow absorption. This has been addressed using site-directed mutagenesis to introduce repulsive charges and steric hindrances at the dimer interface, in order to reduce the tendency of human insulin to self-assemble. This work has led to insulin monomers that have an increased rate of absorption, and thus a produce a preferable postprandial plasma concentration profile [4]. 

Simulation of the structure of the insulin hexamer consisting of six insulin monomers bound together. The amino acid chains are depicted as ribbons, except for the six histidine residues which point toward the center of the molecule. At the very center, bonded to the histidines and locking the whole structure together, is a spherical zinc ion... 

---