Modification of Serum Albumin by High Conversion of Carboxyl to Amino Groups Creates a Potent Inhibitor of Amyloid β-Protein Fibrillogenesis.

Fibrillogenesis of amyloid β-protein (Aβ) have been thought to be implicated in the progression of Alzheimer's disease (AD). Therefore, development of high- efficiency inhibitors is one of the strategies for the prevention and treatment of AD. Serum albumin has been found to capture Aβ monomers through its hydrophobic groove and suppress amyloid formation, but the inhibition efficiency is limited. Inspired by the strong inhibition potency of a basic protein, human lysozyme, we have herein proposed to develop a basified serum albumin by converting carboxyl groups into amino groups with ethylenediamine conjugated on the protein surface. The idea was verified with both bovine and human serum albumins (BSA/HSA). Four basified BSA (BSA-B) preparations with amino modification degrees (MDs) from 8.0 to 41.5 were first synthesized. Extensive biophysical and biological analyses revealed that the inhibition potency significantly increased with increasing the amino MD. BSA-B of the highest MD (41.5), BSA-B4, which had an isoelectric point of 9.7, presented strong inhibition on Aβ42 fibrillation at a concentration as low as 0.5 µM, at which it functioned similarly with 25 µM native BSA to impede 25 µM Aβ fibrillation. Cell viability assays also confirmed that the detoxification of 5 µM BSA-B4 was superior over 25 µM native BSA by increasing cell viability from 60.6% to 96.0%. Fluorescence quenching study unveiled the decrease of the binding affinity between Aβ42 and the hydrophobic pocket region of BSA-B4, while quartz crystal microbalance experiments demonstrated that the binding constant of BSA-B4 to Aβ42 increased nearly five times. Therefore, the increase of electrostatic interactions between BSA-B4 and Aβ42 was the main reason for its high potency. Hence, aminated BSA achieved a conversion of binding way to Aβ from a mainly single-site hydrophobic binding to multi-regional electrostatic interactions. Similar results were obtained with basified HSA preparations on inhibiting the amyloid formation and cytotoxicity. This work has thus provided new insights into the development of more efficient protein-based inhibitors against Aβ fibrillogenesis.