Pre-incubation under High Pressure Accelerates Amyloid Formation from Insulin

Abstract

Recently it has been made clear that some associations or aggregations of abnormally folded proteins, such as -amyloids, are stably present even under physiological condition, and those structures might cause specific diseases called conformation diseases; amyloidosis. [1][2][3][4][5] The present understandings are that a variety of proteins can form amyloids, fibrous aggregations of protein -forms, besides some specific proteins concerned to the diseases.6-10 Basic aspects of amyloid-formation mechanism have been studied by using many types of proteins and various physical and chemical methodologies. [11][12][13][14][15][16][17] Insulin is one of the prototype agents for such fundamental studies [18][19][20][21][22][23][24] and, as a physical perturbation, pressure (high hydrostatic pressure), besides temperature, is utilized in order to control the structural changes. [25][26][27][28][29][30][31][32] In aqueous solutions, insulin exists as a mixture of monomer, dimer and hexamer; mainly in hexamer at pH around 7, in dimer at pH 2.0, and in monomer in 20% acetic acid. [32][33][34] Above room temperature, insulin can form amyloids and the amyloid is performed by nucleation-dependent elongation model. In this model the whole process is divided into three steps; nucleation, elongation and equilibrium and the rate determining step is considered as in the nucleation. 20,21,24 Dissociation to monomers from insulin dimers or hexamers and the following conformational change into modified monomers have to occur in prior to the nucleus formation. Even after amyloid is formed, three disulphide bonds in insulin are present intact without scissions. 35Here we have investigated some fundamental but important points of high pressure effects on -amyloid formation from insulin. EXPERIMENTALThe amyloid formation process was observed by adding fluorescent probe, thioflavine T (ThT: Basic Yellow 1) 20 mM, in the insulin solutions. ThT shows a strong and characteristic fluorescence (ex. 450 nm and em. 480-490 nm), when it is captured in amyloid fibrils, although the details of the mechanisms are not yet fully elucidated. 36,37 Bovine insulin and ThT were purchased from Sigma (St. Louis, Mo. USA). Insulin was dissolved in deionized and filtered water, and pH was adjusted to 2 by adding dilute HCl aqueous solution. The insulin concentration was determined spectrophotometrically (A 1% 276 nm ¼ 1)38 by using Shimadzu spectrophotometer UV-2200. The sample solution containing 5-20 mg/mL of insulin and 20 mM of ThT was portioned and set in a high-pressure cell settled in a fluorescence photometer (Shimadzu RF 5300). The details of the high-pressure equipment have been given elsewhere. 39 The excitation and emission wavelengths were set at 444 and 485 nm, respectively. After fluorescence measurements, the sample solutions were taken out and stained with 3% tungsto(VI) phosphoric acid aqueous solution for electron microscopic observation by JEOL-1220 (80 kV) (Tokyo, Japan). RESULTS AND DISCUSSIONThe time courses of fluorescence change of th...