Self insulin molecule

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. 

Physical properties of the protein structure should be considered in designing strategies to achieve stable formulations because they can often yield clues about which solution environment would be appropriate for stabilization. For example, the insulin molecule is known to self-associate via a nonspecific hydrophobic mechanism66 Stabilizers tested include phenol derivatives, nonionic and ionic surfactants, polypropylene glycol, glycerol, and carbohydrates. The choice of using stabilizers that are amphiphilic in nature to minimize interactions where protein hydrophobic surfaces instigate the instability is founded upon the hydro-phobic effect.19 It has already been mentioned that hydrophobic surfaces prefer... 

Insulin Lispro was the first recombinant fast-acting insulin analogue to gain marketing approval (Table 8.3). It displays an amino acid sequence identical to native human insulin, with one alteration — an inversion of the natural proline lysine sequence found at positions 28 and 29 of the insulin jS-chain. This simple alteration significantly decreased the propensity of individual insulin molecules to self-associate when stored at therapeutic dose concentrations. The dimerization constant for Insulin Lispro is 300 times lower than that exhibited by unmodified human insulin. Structurally, this appears to occur as the change in sequence disrupts the formation of inter-chain hydrophobic interactions critical to self-association. 

Some proteins self-associate in aqueous solution to form oligomers. Insulin, for example, exists in several associated states the zinc hexamer of insulin is a complex of insulin and zinc which dissolves slowly into dimers and eventually monomers following its subcutaneous administration, so giving it long-acting properties. In most cases, however, it is desirable to prevent association such that only monomeric or dimeric forms are present in the formulations and a more rapid absorption is achieved. Recent studies have been directed towards engineering insulin molecules which are not prone to association, " or the prevention of association through the addition of surfactants. Protein self-association is a reversible process, i.e. alteration of the solvent properties can lead to the re-formation of the monomeric native protein. There is an important distinction between this association... 

Self-association of the insulin molecule into oligomers and macromolecular aggregates leads to complications in the development of long-term insulin therapeutic systems and limits the rate of subcutaneous absorptions, a process which... 

Regular crystalline insulin naturally self-associates into a hexam-eric (six insulin molecules) stmcture when injected subcutaneously. Before absorption through a blood capillary can occur, the hexamer must dissociate first to dimers, and then to monomers. This principle is the premise for additives such as protamine and zinc described below, and modification of amino acids for insulin analogs. Lispro, aspart, and glulisine insulins dissociate rapidly to monomers, thus absorption... 

Figure 3. The primary structure of human insulin. The sites and types of mutation in the different analogs with reduced tendency to self-association are indicated. Also shown arc the amino acid residues involved in the association of two insulin molecules into a dimer (black residues) and in the assembly of three dimers and two zinc ion into a Zn-insulin hezamer (shaded residues). The putative sites interacting with the receptor are indicated by arrows. (From Brange cr aJ., 1990, with permission.)...

Insulin Aspart is a second fast-acting engineered human insulin analogue now approved for general medical use. It differs from native human insulin in that the proline residue has been replaced by aspartic acid. This single amino acid substitution also decreases the propensity of individual molecules to self-associate, ensuring that they begin to enter the bloodstream from the site of injection immediately upon administration. 

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. 

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