Abstract
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.
Highlights
A wide range of diseases, which include Alzheimer’s disease, Parkinson’s disease, type 2 diabetes, systemic amyloidosis, and prion-related diseases were associated with the presence of amyloid aggregates in various parts of the body
1) We applied the three protein concentration analysis methods, OD 280, colloidal gold assay, and DC assay and analyzed protein colloids in solution; 2) Based on the difference in the total amount of protein detected for the same solution by the three methods, we quantified the amount of protein on the surface and in the interior of the protein colloids; and 3) Based on a math model we provided, we were able to determine the diameter of the protein colloids
Protein colloids are an important intermediate in amyloid fiber formation
Summary
A wide range of diseases, which include Alzheimer’s disease, Parkinson’s disease, type 2 diabetes, systemic amyloidosis, and prion-related diseases were associated with the presence of amyloid aggregates in various parts of the body. There exist two main steps for amyloid fiber formation. The clinical and biochemical characteristics are distinct, the common feature implicated in the pathogenesis of amyloid-related diseases is self-assembly of normally soluble proteins into amyloid aggregates (dimers, colloids, fibers). This insoluble amyloid fiber cannot be removed from the human body. This phenomenon can exist in tissue, especially in the brain, leading to synaptic dysfunction and cognitive deficits. A way to quantify the diameter of colloids is quite helpful in the understanding of amyloid fiber formation
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