Insulin fibril formation

A prime example of a Refolding model is that of the insulin protofilament (Jimenez et al., 2002). Insulin is a polypeptide hormone composed of two peptide chains of mainly o -helical secondary structure (Fig. 3A Adams et al., 1969). Its chains (21- and 30-amino acids long) are held together by 3 disulfide bonds, 2 interchain and 1 intrachain (Sanger, 1959). These bonds remain intact in the insulin amyloid fibrils of patients with injection amyloidosis (Dische et al., 1988). Fourier transform infrared (FTIR) and circular dichroic (CD) spectroscopy indicate that a conversion to jS-structure accompanies insulin fibril formation (Bouchard et al., 2000). The fibrils also give a cross-jS diffraction pattern (Burke and Rougvie, 1972). 

Nielsen, L., S. Frokjaer, J. Brange, V.N. Uversky, and A.L. Fink, Probing the mechanism of insulin fibril formation with insulin mutants. Biochemistry, 2001.40(28) 8397-409. 

Nielsen L, Khurana R, Coats A, et al. Effect of environmental factors on the kinetics of insulin fibril formation elucidation of the molecular mechanism. Biochemistry 2001 40(20) 6036-6046. 

Insulin fibril formation is particularly important with the advent of infusion pumps to deliver insulin. In these devices, insulin is exposed to elevated temperatures, the presence of hydrophobic surfaces, and shear forces, all factors that increase insulin s tendency to aggregate. These problems can be overcome if the insulin is prepared with phosphate buffer or other additives. Another physical stability problem associated with insulin is adsorption to tubing and other surfaces. This normally occurs if the insulin concentration is less than 5 lU/mL (0.03 mM), and it can be prevented by adding albumin to the dosage form if a dilute insulin solution must be used (34). 

Use of the anionic CP allowed for detection of amyloid fibril formation in both bovine insulin and chicken lysozyme proteins (Fig. 16). The polymer in buffer... 

Fig. 16 (a) Description of the detection of amyloid fibrils in proteins with an anionic conjugated polymer, PTAA. (b) Emission spectra (bottom) of PTAA-Native bovine insulin (filled square) and PTAA-amyloid fibrillar bovine insulin (x). (c) Kinetics of insulin amyloid fibril formation monitored by PTAA fluorescence [29]... 

It appears that the interactions leading to fibril formation result from the monomeric form, and from change in monomer conformation and hydrophilic attraction of the parallel /J-sheet forms. Fibril formation is also encouraged by contact of the insulin solution with hydrophobic surfaces. Contact with gamma-irradiated PVC leads to instability, apparently induced by chemical changes in the insulin. 

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). 

These aggregates contain nonnative structures, for example, intermolecular P-sheet (50), and can cause irreversible changes to the unfolded species, such as precipitation, aggregation, or fibrillation (18,35,43), where precipitation is the macroscopic equivalent of aggregation (40,42). Aggregation is dependent not only on the protein concentration but also on the stress caused by shaking (49,51). The structure of the aggregated proteins can be more or less well defined, and an example of well-defined structures is the formation of insulin fibrils (52,53). 

In the course of our studies on the fibril formation in insulin, we have discovered several interesting possibilities to probe the unusual properties of the fibers. In the first place it is possible to follow the formation of the fibers with ultrasound velocimetry. As can be seen from Fig. 5 the decrease in adiabatic compressibility takes place at the same temperature where the P-structures start to form. The dimension of the fibers was verified by atomic force microscopy. 

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. 

The polymer P2.209 was used as an optical probe in the detection of amyloid fibril formation of bovine insulin (BI) and chicken lysozyme in acetic acid medium. Upon interaction with BI, a drastic increase... 

Figure 22.8 Absorption spectra of (a) 80 nM of polymer 11 (on a monomer basis) in 20 mM sodium phosphate, pH 7.0, (b) in 20 mM sodium phosphate pH 7.0 with 5.0 icM of native bovine insulin (nBI) and (c) in 20 mM sodium phosphate, pH 7.0, with 5.0 fx.M of amyloid fibrils of bovine insulin (fBI). The inset shows the microtiter plate wells containing polymer 3-nBI (left) and polymer 3 fBI (right). Reprinted with permission from K. P. R. Nilsson, A. Herland, P. Hammarstrom and O. Inganas, Conjugated poly electrolytes conformation-sensitive optical probes for detection of amyloid fibril formation. Biochemistry, 44, 3718-3724 (2005). Copyright 2005 American Chemical Society...

Bouchard, M., Zurdo, J., Nettleton, E. J., Dobson, C. M., and Robinson, C. V. (2000). Formation of insulin amyloid fibrils followed by FTIR simultaneously with CD and electron microscopy. Protein Sri. 9, 1960-1967. 

Fig. 15 Schematic drawing of the formation of amyloid fibrils, (a) Monomeric insulin having an a-helical conformation, (b) [i-sheet (arrows) rich oligomers are being formed, (c) Amyloid fibrils having a diameter around 10 nm are being formed, (d) Higher magnification of the intrinsic repetitive (S-pIcatcd sheet structure of the amyloid fibril. The pictures were taken by transmission electron microscopy (TEM)...

Information on the stability of fibrils will be critical for assessing the potential of fibril-coated surfaces for biotechnology applications. Fibrils such as insulin remain strongly bound to the surface after formation and remain on the surface after rinsing with water (Zhu et al., 2002). This is a desirable feature for the construction of new materials, although there may be some instances where surface transfer is required. 

Precipitation of insulin in pumps due to the formation of amorphous particles, crystals or fibrils of insulin can lead to changes in release pattern or to blockage which prevents insulin release. Amorphous or crystalline precipitates can be caused by the leaching of divalent metal contaminants or lowering of pH (due to CO2 diffusion or leaching of acidic substances), but can be prevented. More difficult to solve is the tendency of insulin to form fibrils as illustrated in Fig. f f. T4. 

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