Determination of the Rate Constants of Oligomerization of Amyloid-β Peptides using Fluorescence Quenching of Tetramethylrhodamine

Abstract

Soluble oligomers of amyloid-β (Aβ) play a central role in the pathology of Alzheimer's disease. Here we have determined the rate constants of the monomer-oligomer process of Aβ using a new method based on self-quenching of tetramethylrhodamine (TMR) which is covalently labeled at the N-terminal of Aβ. During aggregation TMR fluorescence is quenched in a time dependent manner in three distinct phases, an early oligomerization phase, a lag phase and a growth phase. Kinetic data of the early phase are consistent with a monomer-dimer-trimer process. The rate and the equilibrium constants differ markedly between Aβ1-42 and Aβ1-40 showing higher oligomerization propensity for A1-42.The association rate constants (∼102 M−1s−1) for both the peptides are slow compared to a diffusion limited process. The rates of oligomerization are altered dramatically with increase of temperature and are highly sensitive to changes in pH especially below pH 7.5. Our results for the first time provide quantitative estimation of the kinetics of monomer-oligomer process of Aβ. The methodologies presented here are simple and therefore can be easily applied to other amyloidogenic proteins.