Topics Covered
Background
Experimental
Sample Preparation
Determination of Particle Size
Results
Background
In 1934, Palmer first described the
separation of ß-Lactoglobulin (ß-Lg) from the whey fraction of bovine milk. This
discovery was made fifty years before the actual structure could be elucidated
through X-ray crystallography.. The majority of these studies centre around heat
induced aggregation since this is of great interest and importance to the
processing of dairy products. Despite the years of research, the specific
function of ß-Lactoglobulin is yet to be completely understood. The purpose of
this work is to provide a study of ß-Lg electrostatic aggregation at low ionic
strength (I) and at pH values below the isoelectric point (pI) where aggregation
occurs at a rate slow enough to be evaluated by quasi-elastic light scattering
(QELS).
Experimental
Sample
Preparation
ß-Lg A&B, purchased from Sigma- Aldrich
and used without further purification, was dissolved in 4.5mM NaCl prepared from
Milli-Q water. The dissolved ß-Lg was then raised to pH 9 in order to
disassociate the protein into its monomeric species. An equal volume of 4.5mM
NaCl was prepared containing the appropriate amount of 0.1M HCl to bring the
combined solutions to the target pH and concentration of 4.2 and 1mg/mL
respectively. Both solutions were filtered (0.2 µm filters, Sartorius AG,
Germany),
and then mixed immediately prior to analysis.
Determination of
Particle Size
Size distributions of the ß-Lg aggregates
were determined using the Malvern
Zetasizer Nano ZS. Correlation times were seven seconds per run and three
runs per measurement. QELS was used to determine size distributions over fifty
minutes.
Results
Figure 1 shows the intensity size
distribution collected during the kinetic experiment at time (t) = 0. As evident
here, the QELS results indicate that, ß-Lg aggregates in two distinct size
populations almost immediately.
Figure 1. Intensity size distribution for
1mg/mL ß-Lg in 4.5mM NaCl at pH 4.2 at time = 0.
The smaller size population has a
hydrodynamic diameter of circa 5.4nm. Using a known mass vs. size relationship
for globular proteins, shown in Equation 1, the molecular weight of the 5.4nm
protein is approximately 35 kDa, consistent with the dimeric form of the ß-Lg
protein.
The kinetic trace for the experiment is
shown in Figure 2, which shows the intensity and Z-Average diameter as a
function of experimental time.
Figure 2. Kinetic trace of 1.0mg/mL ß-Lg
in 4.5mM NaCl at pH 4.2.
The scattering intensity is roughly
proportional to the concentration and the square of the molecular weight, and is
hence quite sensitive to the formation of higher order aggregates. The Z-Average
diameter is the mean diameter of the ensemble of particles, and is derived from
the slope of the linearized form of the correlation function (Cumulants method).
As seen in Figure 2, the formation of the higher order aggregate occurs in the
first 20-25 minutes of the experiment. At longer times, the scattering intensity
is relatively constant, suggesting that dimer-aggregate equilibrium has been
achieved. Stabilization of the higher order aggregate at longer times is also
evident in Figure 3, which shows selected intensity size distributions from the
kinetic experiment.
Figure 3. Selected intensity size
distributions from the ß-Lg kinetic experiment.
A decrease in the relative amount of dimer
was also observed over the time course of the experiment. At the initial
conditions, shown in Figure 1, the dimer concentration accounted for 80% of the
scattering intensity. At the conclusion of the experimental run, the
contribution to scattering intensity by the dimer had stabilized at roughly 10%
of the total (see Figure 4). As seen in Figure 4, an increase in scattering
intensity and size stabilization of the super aggregate could also be seen over
time. The scattering intensity of the aggregate increased from 20% to 90% during
the run, with the size of the aggregate ultimately stabilized at approximately
425nm.
Figure 4. Time dependent intensity size
distributions for ß-Lg in 4.5mM NaCl at pH 4.2.
Note: A complete list of references is
available by referring to the original document.
Source: "Aggregation of β-Lactoglobulin", Application
Note by Malvern Instruments.
For more information on this source please
visit Malvern
Instruments Ltd (UK) or Malvern Instruments
(USA).