Abstract
Protein aggregation is believed to be a significant biological mechanism related to neurodegenerative disease, which makes the early-stage detection of aggregates a major concern. We demonstrated the use of terahertz (THz) time-domain spectroscopy to study protein-water interaction of monomeric and dimeric bovine insulin in aqueous samples. Regulated by changing pH and verified by size-exclusion chromatography and dynamic light scattering, we then measured their concentration-dependent changes in THz absorption between 0.5 and 3.0 THz and quantitatively deduced the extended hydration shell thickness by cubic distribution model and random distribution model. Under a random distribution assumption, the extended hydration thickness is 15.4 ± 0.4 Å for monomeric insulin and 17.5 ± 0.5 Å for dimeric insulin, with the hydration number of 6700 and 11,000, respectively. The hydration number of dimeric insulin is not twice but 1.64 times that of monomeric insulin, further supported by the ratio of solvent-accessible surface area. This "1.64-times" relation probably originates from the structural and conformational changes accompanied with dimerization. Combined with the investigations on insulin samples with different single amino acid mutations, residue B24 is believed to play an important role in the dimerization process. It is demonstrated that THz time-domain spectroscopy is a useful tool and has the sensitivity to provide the hydration information of different protein aggregates at an early stage.