Kinetic modeling and determination of reaction constants of Alzheimer's beta-amyloid fibril extension and dissociation using surface plasmon resonance.
To establish the kinetic model of the extension and dissociation of beta-amyloid fibrils (f(A)beta) in vitro, we analyzed these reactions using a surface plasmon resonance (SPR) biosensor. Sonicated f(A)beta were immobilized on the surface of the SPR sensor chip as seeds. The SPR signal increased linearly as a function of time after amyloid beta-peptides (Abeta) were injected into the f(A)beta-immobilized chips. The extension of f(A)beta was confirmed by atomic force microscopy. When flow cells were washed with running buffer, the SPR signal decreased with time after the extension reaction. The curve fitting resolved the dissociation reaction into the fast exponential and slow linear decay phases. Kinetic analysis of the effect of Abeta/f(A)beta concentrations on the reaction rate indicated that both the extension reaction and the slow linear phase of the dissociation were consistent with a first-order kinetic model; i.e., the extension/dissociation reactions proceed via consecutive association/dissociation of Abeta onto/from the end of existing fibrils. On the basis of this model, the critical monomer concentration ([M](e)) and the equilibrium association constant (K) were calculated, for the first time, to be 20 nM and 5 x 10(7) M(-1), respectively. Alternatively, [M](e) was directly measured as 200 nM, which may represent the equilibrium between the extension reaction and the fast phase of the dissociation. The SPR biosensor is a useful quantitative tool for the kinetic and thermodynamic study of the molecular mechanisms of f9A)beta formation in vitro.
Hasegawa
Kazuhiro
K
Department of Pathology, Fukui Medical University, Fukui 910-1193, Japan. khase@fmsrsa.fukui-med.ac.jp
Ono
Kenjiro
K
Yamada
Masahito
M
Naiki
Hironobu
H
D000544:Alzheimer Disease / Q000378:metabolism* / Q000473:pathology; D016229:Amyloid beta-Peptides / Q000378:metabolism*; D015374:Biosensing Techniques; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008954:Models, Biological; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D013696:Temperature
D016229:Amyloid beta-Peptides; D005456:Fluorescent Dyes