Topics Covered
Background
Experimental
Results
Background
The interactions governing
protein-polyelectrolyte complexation have been the subject of many studies in
the application areas of protein purification, enzyme immobilization,
immunosensing, and bioactive sensors. These studies have also aided in the
understanding of biological systems, for example, DNA-binding proteins, a field
in which the general physical chemistry of the interactions has been
subordinated to studies of storage, replication, and transcription mechanisms.
This application note highlights the use of the Malvern
Zetasizer Nano ZS for characterization of protein-polyelectrolyte complexes
(PPCs).
Experimental
The PPC system examined in this study was
composed of bovine serum albumin (BSA), with an isoelectric point (pI) of 4.8,
and a custom synthesized anionic copolymer of 60%
2-acrylamido-2-methylpropanesulfonate and 40% acrylamide (AMPS60Aam40).
Protein-polyelectrolyte solutions were prepared at a 5:1 mass ratio of protein
to polyelectrolyte in 250 mM NaCl at pH 8.5. Dynamic light scattering
measurements of the filtered solutions (0.22 mm Sartorius) were collected with
the Zetasizer Nano ZS system at 0.2 pH unit intervals from pH 8.5
to pH 4.2. The deconvolution of the measured correlation curve to an intensity
size distribution was accomplished using a non-negative least squares
algorithm.
Results
Figure 1 shows the intensity size
distributions for BSA, AMPS60AAm40, and the complex at pH 8.5. At this pH, the
negative charge on the protein is sufficient to prohibit complexation with the
polyanion. Because of the higher mass concentration and globular nature of the
protein, the unbound polyanion cannot be detected in the presence of
protein.
Figure 1. Intensity size distributions
for BSA, AMPS60Aam40, PAMPS80, and a BSA-AMPS60AAm40 mixture in 250 mM NaCl at
pH 8.5.
The pH dependence of the Z-average diameter
for the protein-polyelectrolyte complex is shown in Figure 2, which indicates a
deviation from baseline at circa pH 5.0. The Z-average size is the intensity
weighted mean diameter, derived from a Cumulants or single exponential fit of
the intensity autocorrelation function. The deviation from linearity shown in
figure 2 is typical of protein-polyelectrolyte mixtures, and allows one to
define the pH of initial complex formation (pHc). At pH 5, the net BSA charge is
negative. As such, the binding of BSA to a polyanion at pH 5.0 suggests that the
protein-polyelectrolye interactions are local, between the negatively charged
polymer and a positive charge patch on the surface of the protein.
Figure 2. pH dependence of the Z-average
diameter of BSA-AMPS60AAm40 in 250 mM NaCl.
Figure 3 shows the intensity size
distributions for the protein-polyelectrolyte complex for pH ¡Ü pHc. As seen in
Figure 3, a bimodal distribution is clearly visible at the onset of
complexation. The faster diffusing mode, with an apparent diameter of 8 nm,
corresponds to free BSA. The slower diffusing mode at circa 75 nm represents the
protein-polymer complex, with an apparent size that is roughly the same as that
of the free polyelectrolyte. As the pH is decreased, the apparent size of the
complex peak shifts to lower values, accompanied by a decrease in the relative
scattering intensity of free BSA. This reduction in the relative scattering
intensity of the free protein suggests that pH reduction leads to an increase in
protein binding and complex formation.
Figure 3. Intensity size distributions
for the BSA-AMPS60AAm40 complex for pH ¡Ü pHc.
Note: A complete set of references can be found
by referring to the source document.
Source: "Characterization of Protein-Polyelectrolyte
Complexes", Application Note by Malvern
Instruments.
For more information on this source please
visit Malvern
Instruments Ltd (UK) or Malvern Instruments
(USA).