ß-Lactoglobulin as a potential carrier for bioactive molecules.

In this study, the interaction and binding behavior of anesthetic tetracaine (TET) with bovine ß-lactoglobulin (LGB) isoform A and a mixture of isoforms A and B were investigated under varying environmental conditions (pH, ionic strength, concentration, LGB-TET complex molar ratio). A wide range of analytical techniques (dynamic light scattering (DLS), electrophoretic mobility, UV-Vis spectroscopy, circular dichroism (CD), quartz crystal microbalance (QCM-D) were used to analyze the physicochemical properties of the complexes in bulk solution and on the surface of gold. The experiments revealed that TET, which is amphiphilic, could bind with LGB not only in the ß-barrel but also onto the surface. The zeta potential of the LGB becomes more positively charged upon interaction with TET due to electrostatic interaction of the amino group present in the TET structure. Changes in the zeta potential values are mostly visible above pH 6 for all tested systems. CD spectra show that interaction with the ligand does not change the secondary structure of the protein. The physicochemical properties of LGB-TET complex were examined in an adsorbed state on a gold surface using the QCM-D method. Additionally, molecular docking was used to evaluate the most likely binding site for TET with LGB.

Adsorption effectiveness of ß-lactoglobulin onto gold surface determined by quartz crystal microbalance.

Bovine ß-lactoglobulin (LGB) is a transport protein that can bind to its structure hydrophobic bioactive molecules. Due to the lack of toxicity, high stability and pH-dependent molecular binding mechanism, lactoglobulin can be used as a carrier of sparingly soluble drugs. Dynamic light scattering has confirmed LGB's tendency to create oligomeric forms. The hydrodynamic diameter of LGB molecules varies from 4 nm to 6 nm in the pH range of 2-10 and ionic strength I = 0.001-0.15 M, which corresponds to the presence of mono or dimeric LGB forms. The LGB zeta potential varies from 26.5 mV to -33.3 mV for I = 0.01 M and from 13.3 mV to -16 mV for I = 0.15 M in the pH range of 2-10. The isoelectric point is at pH 4.8. As a result of strong surface charge compensation, the maximum effective ionization degree of the LGB molecule is 35% for ionic strength I = 0.01 M and 22% for I = 0.15 M. The effectiveness of adsorption is linked with the properties of the protein, as well as those of the adsorption surface. The functionalization of gold surfaces with ß-lactoglobulin (LGB) was studied using a quartz crystal microbalance with energy dissipation monitoring (QCM-D). The effectiveness of LGB adsorption correlates strongly with a charge of gold surface and the zeta potential of the molecule. The greatest value of the adsorbed mass was observed in the pH range in which LGB has a positive zeta potential values, below pH 4.8. This observation shows that electrostatic interactions play a dominant role in LGB adsorption on gold surfaces. Based on the adsorbed mass, protein orientation on gold surfaces was determined. The preferential side-on orientation of LGB molecules observed in the adsorption layer is consistent with the direction of the molecule dipole momentum determined by molecular dynamics simulations of the protein (MD). The use of the QCM-D method also allowed us to determine the effectiveness of adsorption of LGB on gold surface. Knowing the mechanism of LGB adsorption is significant importance for determining the optimum conditions for immobilizing this protein on solid surfaces. As ß-lactoglobulin is a protein that binds various ligands, the binding properties of immobilized ß-lactoglobulin can be used to design controlled protein structures for biomedical applications.

Investigation of high pressure effect on the structure and adsorption of ß-lactoglobulin.

ß-Lactoglobulin, being one of the principal whey protein, is of huge importance to the food industry. Temperature/pressure effects on this small protein has been extensively studied by industry. To characterize biochemical properties of ß-lactoglobulin after or during pressurization, a wide range of methods have been used thus far. In this study, for the first time, the pressure-induced conformation of ß-lactoglobulin in the crystal state was determined, at pressure 430 MPa. Changes observed in the high pressure structure correlate with the physico-chemical properties of pressure-treated ß-lactoglobulin obtained from dynamic light scattering, electrophoretic mobility and quartz crystal microbalance with dissipation monitoring measurements. A comparison between the ß-lactoglobulin structures determined at both high and ambient pressure contrasts the stable nature of the protein core and adjacent loop fragments. At high pressure the ß-lactoglobulin structure presents early signs of dimer dissociation, charge and conformational changes characteristic for initial unfolded intermediate as well as a significant modification of the binding pocket volume. Those observations are supported by changes in zeta potential values and results in increase affinity of the ß-lactoglobulin adsorption onto gold surface. Observed pressure-induced structural modifications were previously suggested as an important factor contributing to ß-lactoglobulin denaturation process. Presented studies provide detailed analysis of pressure-associated structural changes influencing ß-lactoglobulin conformation and consequently its adsorption.