A Novel Method To Study Interaction Proteins Of Oligomeric Amyloid-β

Oligomeric amyloid-β(oAβ) plays important roles during the development of Alzheimer's disease (AD). However, the uniform oAβis difficult to achieve during the research. This paper proposed a novel method to study the interactions proteins of oAβby Chromatography, in which the oAβwere achieved utilizing molecular clusters that was immobilized on Sepharose CL-4B. Firstly, Asp and Asp12 were selected as examples of molecular clusters and immobilized on Sepharose CL-4B. Then Aβmonomers were coupled on -COOH of Asp/Asp12. At last two adsorbents, named Aβ2- and Aβ13-Sepharose CL-4B, were formed by modifying pH from 11.5 to 6.3. During the synthetic process, efficiency of reactions was controlled to obtain homogeneous. After that, interaction proteins were adsorbed and separated from brain proteins of 2- and 12-month old mice through the two adsorbents. This method might be employed to provide an effective approach for the research of the pathology of oAβ.Experimental results showed:(1) Effect of pH (8-12) and reaction time (2-24 h) on density of coupled Asp were investigated firstly. The optimized reactive condition was pH 11 and 3 h, regarding of the high immobilized efficiency and avoiding the interaction of molecular cluster.(2) For the option of the space arm, bromoacetamide was selected as the linker to immobilize Aβ. And after optimization, pH11.5,12 h and Aβconcentration as twice over the former active group concentration was chosen.(3) Immobilized oAβwas detected by thioflavinT. It was found that the fluorescence intensity of Aβ13- Sepharose CL-4B was stronger than Aβ2- Sepharose CL-4B, while there was no signal for the control. So it was proved that oAP are achieved by immobilized method.(4) Aβ2- and Aβ13-Sepharose CL-4B both could interact with kinds of brain proteins of mouse. And with the development of age from 2- to 12-month, the total adsorption capacity of Aβ2-and Aβ13-Sepharose CL-4B increased 1.75 and 2.2 times, respectively. The adsorbates were less than 1% of the total mouse brain proteins.(5) SDS-PAGE electrophoresis showed both Aβ13 and Aβ2 adsobed kinds of brain proteins. However, the different of proteins adsorbed by two adsorbents is unobservable by single-dimensional electrophoresis.(6) Comparison of Aβ13 interaction proteins with 2D-PAGE electrophoresis showed that all proteins, except for 3 of them, were differential between 2- and 12-month mice. Moreover, the adsorption capacities of the identical proteins in 12-month old mouse were higher than that in 2-month old.