Protein self-assembly and formation of amyloid fibers is an early event of numerous human diseases. Continuous aggregation of amyloid fibers in vitro produces biogels, which led us to suspect that amyloid plaques and neurofibrillary tangles in Alzheimer’s disease are of biogels in nature. We applied atomic force microscopy, size exclusion chromatography, and differential scanning calorimetry to elucidate the gel’s structure, kinetics of gel formation, and melting point. We found that (1) lysozyme gelation occurs when the protein concentration is above 5 mg/mL; (2) nonfibrous protein concentration decreases and plateaus after three days of gel synthesis reaction; (3) colloidal lysozyme aggregates are detectable by both atomic force microscopy (AFM) and fast protein liquid chromatography (FPLC); (4) the gels are a three-dimensional (3D) network crosslinked by fibers coiling around each other; (5) the gels have a high melting point at around around 110 °C, which is weakly dependent on protein concentration; (6) the gels are conductive under an electric field, and (7) they form faster in the presence than in the absence of salt in the reaction buffer. The potential role of the gels formed by amyloid fibers in amyloidosis, particularly in Alzheimer’s disease was thoroughly discussed, as gels with increased viscosity, are known to restrict bulk flow and then circulation of ions and molecules.
A new method for the analysis of protein colloidal diameter has been developed using three existing protein concentration quantification techniques, absorption at 280 nm, colloidal gold assay, and DC protein assay. Protein colloids are formed in the process of aggregation and are thought to be intermediates in protein self-assembly and formation of amyloid fiber. Deposition of the protein fibers in tissues leads to numerous human diseases including Alzheimer's. Lysozyme was incubated at pH 2.0, 55˚C, an environment conducive to amyloid fiber formation. The protein colloids present in the supernatant of the samples after centrifugation were studied over a time course of 30 days. The OD 280 assay detects total protein concentration based on absorption of radiation in the near UV. The colloidal gold assay and DC protein assay only measure colloidal sphere surface protein concentration. Due to the surface plasmon resonance, the light absorption spectrum changes when proteins bind to colloidal gold particles. Using the measured protein concentration on the surface of protein colloids along with the total measured protein concentration in the entire protein colloidal spheres, an interior protein concentration for all colloids is obtained. The protein colloidal sphere size can be calculated by using the ratio between the interior protein concentration and total protein concentration. Results indicate that the colloidal gold assay, DC protein assay, and OD 280 assay can be used to quantify the size of the protein colloids. The colloidal gold assay and DC protein assay are both independently effective in analysis of surface protein concentration in protein colloids. The DC protein assay was found to be much quicker in data production as it only requires 15 minutes of incubation time. The DC protein assay was also more reliable than the colloidal gold assay in accuracy and precision of results.
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