Zeta-potential and particle size analysis of human amelogenins

Abstract

The developing enamel matrix is a highly dynamic system mainly composed of the full-length amelogenin and its proteolytic cleavage products. In this study, size, zeta-potential, and the isoelectric points of nanoparticles of the recombinant full-length human amelogenin (rH174) and two proteolytic products (rH163 and rH146) were analyzed by dynamic light-scattering and electrokinetic measurements. We tested the hypothesis that zeta-potential may be used as a control parameter in directing the self-assembly of amelogenins. Extensive aggregation of amelogenin molecules with the particle size reaching about one micron occurred at a mildly acidic to neutral pH, and coincided with the red shift of the internal fluorescence. Zeta-potential was between +/- 15 mV in the same pH range, indicating that amelogenin aggregation occurred when surface potentials were minimal. This suggests that electrostatic interactions may be another crucial factor, aside from hydrophobic interaction, in the aggregation and hierarchical assembly of spherical particles of amelogenins into supramolecular structures of a higher order.

Figure 1.

Functions of the average particle size and ζ-potential on pH for the aqueous suspensions of rH174 (a), rH163 (b), and rH146 (c) (N = 15, 6, 4, respectively), including the amino acid sequences thereof (d). The graphs were made by overlapping the results of analyses of proteins derived from different recombinant batches, and each datapoint was obtained as an average of over 100 acquisitions. The bolded amino acids in the sequences of rH174, rH163, and rH146 represent the residues with titratable side-chains.

Figure 2.

Intrinsic fluorescence spectra of aqueous suspensions of rH174 at different pHs (λ = 280 nm).

Figure 3.

AFM images of rH174 aggregates dispersed in water and deposited on glass, at pHs 5.5 (a) and 7.6 (b).