Binding of trypsin to fibrillar amyloid beta-protein

We have recently reported that fibrillar amyloid beta-protein (Aβ) inhibits the proteolytic activity of trypsin and high molecular weight bovine brain protease. We report here that trypsin binds to fibrillar Aβ (fAβ) and the resulting complex of trypsin/fAβ is sodium dodecyl sulfate (SDS)-stable. Electron microscopic analysis confirmed the binding of trypsin on the fibrils of both Aβ 1–40 and Aβ 1–42. SDS-polyacrylamide gel electrophoresis (PAGE) of fAβ sample incubated in the presence of trypsin showed that major amount of trypsin was associated with fAβ that did not enter the gel. The presence of trypsin in this protein complex was confirmed by Western blotting after its elution from the gel. Kinetic studies showed that the binding of trypsin to fibrillar Aβ was dependent on the degree of Aβ fibrillization and on the concentration of fAβ. However, the trypsin binding to Aβ oligomers did not affect the fibril growth. The maximum binding (Bmax) of trypsin to fAβ 1–40 and fAβ 1–42 was 36 pmol and 40 pmol, and dissociation constant (Kd) was 18.31 μM and 20 μM respectively. Similar to fAβ, trypsin could also bind to fibrillar amylin. This binding was dependent on the concentration of fibrillar amylin. Under similar conditions, bovine serum albumin did not bind to fibrillar Aβ. These results suggest that fAβ and fibrillar amylin have strong affinities for trypsin, and chelation of proteases by abnormal aggregated proteins may be a general mechanism for inflicting pathological conditions in various diseases.