Fibrous aggregates of amyloid-β (Aβ) are the main component of plaque lining the cerebrovascular system in cerebral amyloid angiopathy. As the main Aβ isoform in vascular deposits, Aβ40 is a valuable target in the study of cerebral amyloid angiopathy. However, the slow process of Aβ40 aggregation in vitro is the bottleneck in the search for Aβ targeting molecules. Therefore, the method of accelerating the accumulation of Aβ40 in vitro has become a top priority. Experts used various in vitro methods to evaluate the aggregation ability of biological buffer bicine. The data shows that bicine promotes the aggregation of Aβ40 with high speed and reproducibility, resulting in a mixture of aggregates with significantly enriched β-sheet fibril formation and toxicity.

The concentration and time-dependent effects of bicine on the formation of Aβ40 peptide fibrils were first examined by ThT fluorescence measurement. The direct binding of ThT to the β-sheet structure of amyloid will result in characteristic changes in fluorescence intensity [9]. To analyze the tendency of amyloid fibril formation with increasing buffer concentration, we incubated the monomeric Aβ40 peptide (50 μM) with a series of concentrations of bicine (78.130 μM to 20.000 mM) for 2 days at 37°C. It was found that Bicine can effectively promote the formation of β-sheet-rich fibrils in a short period of time (EC50=837 nM) (Figure 1).

The fluorescence intensity increases in a concentration-dependent manner. Since 20 mM is the highest concentration that did not show cytotoxicity (S1 graph), 20 mM dichlorooctane was used in the following experiments. Subsequently, a time-dependent ThT measurement was performed to check the fibrillation rate induced by Shuangxin (Figure 1B). Aβ40 monomer was incubated in bicine (20 mM) or control PBS solution at 37°C for 2, 4, and 7 days, and the ThT fluorescence intensity was scanned. We found that 2 days of incubation in bicine was sufficient to effectively form significantly higher levels of Aβ40 fibrils than those incubated in PBS of the same duration. It was shown that Bicine continuously induced fibrillation for 7 days, while no significant increase in fiber level was observed in the PBS solution.

Figure 1. Fibrillation dynamics of Aβ(1-40) aggregation in Bicine solution.

In order to confirm whether incubation in bicine for 2 days will produce enough mature fibrils, we performed far-ultraviolet CD and TEM. It was found that in the presence of bicine (20 mM), Aβ40 showed a single negative band after 2 days of incubation, with a minimum value of 216 nm, which is a typical CD spectrum of a well-defined β-sheet structure [21]. In addition, a large number of elongated amyloid fibrils of 40 samples were observed in Aβ by TEM and incubated in bicine, while fibril formation was less in PBS. Overall, these results indicate that incubation in bicine for 2 days is sufficient to promote the formation of β-sheet-rich Aβ40 fibrils with an elongated morphology in vitro.

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It can be seen from the results that the aggregation conditions using bicine buffer can quickly obtain toxic β-sheet-rich Aβ40 aggregates, and no side effects of bicine were observed during the acquisition of Aβ40 fibrils in this study. You will definitely ask, where are the high-quality bicine manufacturers available? Desheng Biochemical is recommended here, which can not only provide special testing services, such as bioburden, heavy metal protein, enzyme activity testing, etc. In addition, we also provide a variety of packaging options, welcome to consult and order!