Biomedical Engineering Reference
In-Depth Information
solution volume may affect the protein corona layer. The other drawback is that
during the centrifugation high molecular weight proteins as well as protein
agglomerates also sediment at the bottom of tube which can be falsely identified
as protein corona [
8
]. Therefore, for more accuracy, centrifugation should be
employed in conjunction with other methods such as gel filtration using size-
exclusion chromatography, magnetic separation, and microfiltration.
4.2 Circular Dichroism
Protein secondary structures (like
-sheet) have their own characteristic
circular dichroism (CD) spectra in the ultraviolet (UV) region. CD method has been
widely used for monitoring conformational changes induced by protein-NP
interactions [
9
]. Since NPs are not chiral, they do not interfere with the protein
CD spectra. The CD signal reflects an average of the entire molecular population.
Thus, while CD can determine that a protein contains about 50 %
α
-helix and
β
-helix, it cannot
determine which specific residues are involved in the alpha-helical portion. The CD
spectrum of a protein in the “near-UV” spectral region (250-350 nm) can be
sensitive to certain aspects of tertiary structure. At these wavelengths, the
chromophores are the aromatic amino acids and disulfide bonds, and the CD signals
they produce are sensitive to the overall tertiary structure of the protein. Signals in
the region from 250 to 270 nm are attributable to phenylalanine residues, signals
from 270 to 290 nm are attributable to tyrosine, and those from 280 to 300 nm are
attributable to tryptophan. Disulfide bonds give rise to broad weak signals through-
out the near-UV spectrum. This kind of spectrum can be sensitive to small changes
in tertiary structure due to protein-protein interactions and/or changes in solvent
conditions. Although CD cannot be applied on protein complex mixture, it can
provide useful information on protein structure changes adsorbed on NP surface.
Recently, the teams of Joint Research Centre, Institute for Health and Consumer
Protection (JRC-IHCP), have developed a new method to detect and measure
changes to the structure and stability of proteins interacting with nanoparticles. In
collaboration with researchers at the diamond synchrotron radiation source in the
UK, they have shown that using synchrotron radiation-based circular dichroism
(SRCD) spectroscopy, it is possible to measure, with unprecedented sensitivity, the
alterations that proteins undergo when attaching to nanoparticles [
10
].
This technique allows the measurement of critical parameters related to
protein-nanoparticle interactions using only a few micrograms of proteins. It will
provide much needed data on the relative stability of key biological proteins and aid
in understanding and predicting the potential toxicology of nanomaterials; eventu-
ally, it may contribute to the design of the next generation of nontoxic nanoparticle-
based drug delivery systems.
Su et al. [
11
] investigated the conformational changes of different peptides on
the surface of carbon nanotube at different pH values. As it is shown in Fig.
4.1
,
beta-sheetlike configuration of UW-1 at low pH changes to a random coil at neutral
α
