The Role Of Metal Ions On The Formation Of Amyloid Fibrils Of Bovine Insulin

One of the most intriguing new issues of medical pathology,in the post-genomic era,is represented by "conformational diseases".In the category of conformational diseases an eminent role is played by diseases caused by intracellular or extracellular protein aggregation.Protein aggregation is a negative event in nature and generally represents a failure in the accomplishment of the correct spatial arrangement necessary to a functional protein.When the exposure of hydrophobic surface of protein appears, proteins aggregate to minimize thermodynamically unfavorable interactions between solvent and exposed hydrophobic residues of proteins.Hydrophobic interaction is considered to be the major driving force for both protein folding and aggregation.In certain cases the protein aggregation occurs outside the cytoplasm,the aggregate has a fibrillar shape that in human pathology is described as amyloid deposit.Some very important neurodegenerative diseases such as Alzheimer's disease and spongiform encephalopathy are associated with the deposition of amyloid fibrils. A number of non-disease-associated proteins have also been found to be capable of forming amyloid aggregates under appropriate conditions,such as insulin and albumin. Formation of amyloid fibril is an intrinsic propensity of all the proteins,not depending on their sequences and three-dimensional structures.It is of great significance to explore the circumstances under which a protein forms amyloids favorably,leading to a precise elucidation of the molecular mechanism of amyloid fibril formation.Insulin which is secreted by the isletβ-cell in animal functions as a hormone to regulate the glucose metallization.It is a small protein consisting of 2-chains(A chain and B chain) with a largely a-helical structure.These two chains are linked by two disulfide bonds and there is also an intra-chain disulfide in the A chain.There is a high comparability in bovine insulin and human insulin.Insulin is one of the peptides that are highly prone to form amyloid fibrils,although no evidence has been found the insulin aggregates in vivo are related to a disease.However,insulin fibrils pose a variety of problems in biomedical and biotechnological applications.Amyloid deposits of insulin have been observed in pharmaceutical products and in patients with diabetes after repeated injection of this hormone.So far,a number of studies have been focused on the amyloid fibrillation of insulin. These works include identifying residues critical for aggregation,searching for factors that lead to inhibition or enhancement of fibrillation,and characterizing the hierarchical assemblies,such as oligomers,protofibrils and fibrils in the amyloid pathway.The conversion of native polypeptide into amyloid fibril is strongly dependent on the physical and chemical environment.In the present study,we have examined the effects of metal ions on the amyloid fibrillation of bovine insulin.The growth and morphology of insulin fibril were monitored and characterized by thioflavin T fluorescence,ANS binding,circular dichroism,Raman spectroscopy,Congo Red assay and correlation analysis.All metal ions except Fe³⁺ tested herein accelerated the formation of insulin fibril.The growth of amyloid fibril was accompanied with a partially exposure of the hydrophobic interior of insulin.Correlation analyses showed that the acceleration of insulin fibrillation was related to free energy of hydration, radius of hydration and electrostatic contribution of the metal ions.The breakage of disulfide linkages and the formation of free thio groups were observed in the process of the insulin fibril formation.Potential implications of these findings to the amyloid fibrillation of insulin and the interaction of insulin and metal ions are exploited and discussed.