The important protein hormone insulin, responsible for different kind of functions in our body but mainly storage of nutrients, has for a long time been used for treatment of diabetic patients. This important protein is both physically and chemically unstable. Especially during production where the insulin protein is exposed to unnatural environmental conditions such as acidic pH has this been causing problems, since huge volumes of the product go to waste.  In the human body the environment for the protein is tolerable with normal body temperature and the right pH, but when the protein is commercially synthesised the environmental conditions are not ultimate. What happens during these unfavourable conditions is that the insulin starts to fibrillate. Meaning that linear, biologically inactive aggregates are formed. If then under these conditions such as high temperature and acidic pH, the insulin comes in contact with hydrophobic surfaces then the fibrillation of the protein goes even faster. In the following experiment I am going to investigate if the experiments and conclusions done before, where different kind of additives to insulin solutions have been used to enhance the amyloid fibrillation of insulin, are as effective as it has been proposed and I am going to prove that the presence of hydrophobic surfaces, such as coated silicon surfaces or glass and addition of preformed fibrils, so called seeds, increase amyloid fibrillation of the insulin protein under certain conditions, in comparison with the normal fibrillation under the same conditions. 

Insulin molecule and amyloid fibril formation  


The hormone insulin is a protein consisting of 51 amino acids, divided on two chains. One A – chain containing 21 amino acids and one B- chain containing 30 amino acids. It is produced in the pancreas and is catabolized in the liver, kidney and placenta. This important hormone is mainly promoting storage of ingested nutrients and fat but it is also responsible for many other functions, like promoting protein synthesis in the muscles.  

Effective treatment of diabetic patients with insulin have been taking place for a long time now, and constant improvement and optimization of both therapeutic use and production of insulin is therefore of the greatest importance. 

The integrity of insulin is strongly dependent on environmental conditions making it both physically and chemically unstable.This means that simple changes like the pH, effects the state of the protein. In solution it exists in three states: hexameric, dimeric and monomeric and for example a small concentration of acid, such as HCl, makes the monomeric state predominant.  

A major stability problem is amyloid-like fibril formation, a process where linear, biologically inactive, aggregates are formed, by interactions between non native insulin molecules. In patients with type II diabetes amyloid deposits have been seen after repeated injections and in normal aging, but still the largest problem with aggregation of insulin is during production where large quantity of the insulin needs to be thrown away during commercial isolation and the purification steps, where many of them involve pH in the range of 1-3. Fibril formation consists of three reaction steps. The first one is nucleation, where several misfolded protein monomers form an organized structure so called the nucleus. This nucleus is a precursor for the second reaction. In the second reaction elongation of the nucleus is taking place, meaning more monomers are added and the nucleus is growing into fibrils. The last reaction is floccule formation or so called precipitation. 

Since the first step in fibril formation is dependent on a temperature above ambient it is possible to study the formation of fibrils as a kinetic experiment, taking samples of incubated insulin over time and comparing the amount of formed fibrils. By studying the kinetics of the fibril formation of insulin, researchers are hoping to reveal important information making it possible to prevent this process. 

It has been proposed that the aggregation of insulin is hydrophobic in nature. That the initial step is formation of conformationally changed monomers where the hydrophobic face of the insulin molecule that is normally buried in the diamer and hexamer, becomes exposed to solvent. And it has been proved that insulin forms fibrils more easily in the presence of hydrophobic surfaces.