Mechanism of lysozyme adsorption onto gold surface determined by quartz crystal microbalance and surface plasmon resonance.

In this study, the physicochemical characterization of lysozyme adsorbed on gold was investigated. Through the use of MP-SPR it was possible to establish that the orientation of molecules changes from side-on to between or end-on with increasing surface coverage. The data confirms that the process of adsorption is driven primarily by electrostatic interactions but also by hydrophobic forces. MP-SPR data was compared with the Random Sequential Adsorption model for a molecule with an ellipsoidal shape. Contact angle measurements showed that higher surface coverage also translates in more hydrophilic properties of obtained lysozyme layer. Comparison of CD and PM-IRRAS spectra in solution and adsorbed state respectively showed changes in the secondary structures of lysozyme. These changes are dependent on pH, but fundamentally they go in the direction of the increase of ß-turn/random content with a simultaneous decrease in ß-sheet fraction, which suggests that aggregation is not occurring. The combination of MP-SPR and QCM-D measurements allowed the estimation of the number of water molecules associated with the lysozymes films. It has been observed that hydration decreases from 70% in pH = 4 to 30% in pH = 11. This data indicates that hydration is driven mainly by the degree of protonation of lysozyme molecules.

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.

Lysozyme encapsulated gold nanoclusters: effects of cluster synthesis on natural protein characteristics.

The study of gold nanoclusters (AuNCs) has seen much interest in recent history due to their unique fluorescence properties and environmentally friendly synthesis method using proteins as a growth scaffold. The differences in the physicochemical properties of lysozyme encapsulated AuNCs in comparison to natural lysozyme are characterised in order to determine the effects AuNCs have on natural protein behaviour. The hydrodynamic radius (dynamic light scattering), light absorbance (UV-Vis), electrophoretic mobility, relative density, dynamic viscosity, adsorption (quartz crystal microbalance) and circular dichroism (CD) characteristics of the molecules were studied. It was found that lysozyme forms small dimer/trimer aggregates upon the synthesis of AuNCs within the protein. The diameter of Ly-AuNCs was found to be 8.0 nm across a pH range of 2-11 indicating dimer formation, but larger aggregates with diameters >20 nm were formed between pH 3 and 6. The formation of larger aggregates limits the use of Ly-AuNCs as a fluorescent probe in this pH range. A large shift in the protein's isoelectric point was also observed, shifting from 11.0 to 4.0 upon AuNC synthesis. This resulted in major changes to the adsorption characteristics of lysozyme, observed using a QCM. A monolayer of 8 nm was seen for Ly-AuNCs at pH 4, offering further evidence that the proteins form small aggregates, unlike the natural monomer form of lysozyme. The adsorption of Ly-AuNCs was seen to decrease as pH was increased; this is in major contrast to the lysozyme adsorption behaviour. A decrease in the α-helix content was observed from 25% in natural lysozyme to 1% in Ly-AuNCs. This coincided with an increase in the ß-sheet content after AuNC synthesis indicating that the natural structure of lysozyme was lost. The formation of protein dimers, the change in the protein surface charge from positive to negative, and secondary structure alteration caused by the AuNC synthesis must be considered before attempting to utilise Ly-AuNCs as in vivo probes.

Human serum albumin encapsulated gold nanoclusters: effects of cluster synthesis on natural protein characteristics.

The differences in the physiochemical properties between native Human Serum Albumin (HSA) and HSA encapsulated gold nanoclusters (HSA-AuNCs) are characterised. The light absorbance (UV-Vis), electrophoretic mobility, dynamic viscosity, density, hydrodynamic radius (DLS), absorption (QCM) and chemical bonding (XPS) characteristics of the molecules were studied. The UV-Vis and DLS data show the formation of large aggregates for HSA-AuNCs between pH 4-6 which is not observed for native HSA. This observation was further supported by QCM measurements showing a large increase in mass adsorbed at pH 6 between HSA and HSA-AuNCs. The DLS data also revealed a hydrodynamic diameter of 12 nm for HSA-AuNCs, nearly double that of 7 nm for native HSA at pH higher than 6, suggesting the formation of compact HSA-AuNCs dimers. The electrophoretic mobility data for both HSA-AuNCs and HSA were converted to zeta potentials. The zeta potential of HSA-AuNCs was seen to be more negative between pH 6-12, suggesting that the protein surface interacts with unreacted gold salt anions. Measurements of density and viscosity were also found to be in agreement with previous data suggesting that HSA-AuNCs form aggregates. XPS data also suggest that not all reactants are used up during the HSA-AuNC synthesis and positive side chains play a part in the initial synthesis stages. It was concluded that HSA-AuNCs most likely form dimers at natural and high pH. Between pH 4-6 HSA-AuNCs form very large aggregates limiting their use as fluorescent probes in this pH range. It was also found that the native characteristics of HSA are altered upon HSA-AuNC synthesis which needs to be taken into consideration when applying HSA-AuNCs as fluorescent probes in all fluorescent imaging and sensing.

Cellular stress induced by photodynamic reaction with CoTPPS and MnTMPyPCl5 in combination with electroporation in human colon adenocarcinoma cell lines (LoVo and LoVoDX).

Two porphyrins, CoTPPS and MnTMPyPCl5, were tested for their photodynamic activity and potential novel use in a therapy of human cancers. We investigated an effect of photodynamic reaction (PDR), electroporation (EP) and their combination (electro-photodynamic reaction [EP-PDR]) on human colon adenocarcinoma cell lines (LoVo and resistant to doxorubicin LoVoDX), human breast adenocarcinoma (wild type MCF-7/WT and resistant to doxorubicin MCF-7/DOX), and human melanoma (Me45). The efficiency of macromolecules transport was examined with cytofluorymetry by assessing the degree of propidium iodide (PI) penetration. Additionally, cellular ultrastructure after EP was evaluated. We determined cyto- and photo-cytotoxic effect on the cells viability (MTT assay) after standard PDR and PDR combined with EP. Intracellular distribution and mitochondrial colocalization of both porphyrins was also performed. The experiments proved that both complexes exhibit desirable photodynamic properties on LoVo LoVoDX cells, and EP effectively supports photodynamic method in this type of cancer. The application of EP provided shorter time of incubation (only 10 min) and enhanced effect of applied therapy. The porphyrins did not affect the MCF-7 and Me45 cell lines.

Protonation of lysozymes and its consequences for the adsorption onto a mica surface.

Several physicochemical properties of chicken egg white lysozyme (LSZ) in electrolyte solutions were determined. The hydrodynamic diameter of LSZ at an ionic strength of 0.15 M was found to be 4.0 nm. Using the determined parameters, the number of uncompensated (electrokinetic) charges, N(c), on the molecule surface was calculated from the electrophoretic mobility data. It was found that the N(c) = 2.8 at pH = 3.0 and an ionic strength of I = 0.15 M. At the lower ionic strength, I = 1 × 10(-3) M, this positive charge increased to N(c) = 5.6 at a pH = 3.0 The physicochemical characteristics were supplemented by the dynamic viscosity measurements. The intrinsic viscosity and the hydrodynamic diameter results were compared with theoretical predictions from Brenner's model. Using this approach, it was found that the effective molecule length of LSZ is equal to L(ef) = 5.6 nm. Additional information on the LSZ adsorbed films was obtained by the contact angle measurements. The notably large contact angles were measured on LSZ films formed under the conditions where both the LSZ and the mica were oppositely charged. The higher the positive zeta potential of LSZ, the greater the contact angle measured, which indicates that LSZ affinity for the adsorption on mica increases with its uncompensated charge. The adsorption dependence on the zeta potential of LSZ was explained, assuming a roughly uniform distribution of the net charge on the molecule surface. This assumption is supported by the results of depositing negatively charged, fluorescent latex particles onto the mica surface, which had been modified by LSZ adsorption. The highest latex coverage was formed on mica surfaces that had first been coated with LSZ solutions of lower pH, as a result of the increasing charge of LSZ monolayers in this condition.

Physico-chemical characterization of bovine serum albumin in solution and as deposited on surfaces.

The interactions between proteins and solid surfaces are important for the formation of biocompatible materials. In this study, the physicochemical properties of Bovin serum albumin (BSA) in solution and on a solid surface were studied. The zeta potential and number of uncompensated charges on BSA surfaces were determined from electrophoretic mobility measurements. The dynamic viscosity was also measured to determine BSA conformations in solution, and the data were converted to the effective length L(ef) of the BSA molecule. The length of a BSA molecule was measured to be 8.3 nm in the compact state (N form at pH 4-9) and 26.7 nm in the extended state (F-form). This study demonstrates that the relationship between the hydrodynamic radius, dynamic viscosity and electrophoretic mobility can provide information about the shape and conformation of biopolymer in solution. The contact angle measurements and deposition of fluorescent latex particles were used to characterise BSA monolayers on a mica surface, which were produced by controlled adsorption under diffusion transport. The results suggest that the distribution of charge across a BSA molecule is heterogeneous as evidenced by the presence of positive and negative patches. The maximum contact angle was observed under conditions in which both BSA and mica were oppositely charged. A higher positive zeta potential of BSA was observed to correlate with a higher contact angle. However, at a higher negative zeta potential, BSA exhibited a lower binding affinity. The charge distribution across BSA monolayers was also studied via the colloidal deposition method using negatively charged fluorescent latex particles. Unexpectedly, the fluorescent latex particles adsorbed onto BSA monolayers, even when the effective zeta potential of BSA was negative. This phenomenon may originate from the heterogeneous charge distribution across BSA molecules.

Structure of poly(acrylic acid) in electrolyte solutions determined from simulations and viscosity measurements.

In this work, the structure of poly(acrylic acid) (PAA) molecules in electrolyte solutions obtained from molecular dynamic simulations was compared with experimental data derived from dynamic light scattering (PCS), dynamic viscosity, and electrophoretic measurements. Simulations and measurements were carried out for polymer having a molecular weight of 12 kD for various ionic strengths of the supporting electrolyte (NaCl). The effect of the ionization degree of the polymer, regulated by the change in the pH of the solution in the range 4-9 units, was also studied systematically. It was predicted from theoretical simulations that, for low electrolyte concentration (10(-3) M) and pH = 9 (full nominal ionization of PAA), the molecule assumed the shape of a flexible rod having the effective length L(ef) = 21 nm, compared to the contour length L(ext) = 41 nm predicted for a fully extended polymer chain. For an electrolyte concentration of 0.15 M, it was predicted that L(ef) = 10.5 nm. For a lower ionization degree, a significant folding of the molecule was predicted, which assumed the shape of a sphere having the radius of 2 nm. These theoretical predictions were compared with PCS experimental measurements of the diffusion coefficient of the molecule, which allowed one to calculate its hydrodynamic radius R(H). It was found that R(H) varied between 6.6 nm for low ionic strength (pH = 9) and 5.8 nm for higher ionic strength (pH = 4). The R(H) values for pH = 9 were in a good agreement with theoretical predictions of particle shape, approximated by prolate spheroids, bent to various forms. On the other hand, a significant deviation from the theoretical shape predictions occurring at pH = 4 was interpreted in terms of the chain hydration effect neglected in simulations. To obtain additional shape information, the dynamic viscosity of polyelectrolyte solutions was measured using a capillary viscometer. It was found that, after considering the correction for hydration, the experimental results were in a good agreement with the Brenner's viscosity theory for prolate spheroid suspensions. The effective lengths derived from viscosity measurements using this theory were in good agreement with values predicted from the molecular dynamic simulations.

Characterization of polyelectrolyte multilayers by the streaming potential method.

Polyelectrolyte multilayer adsorption on mica was studied by the streaming potential method in the parallel-plate channel setup. The technique was calibrated by performing model measurements of streaming potential by using monodisperse latex particles. Two types of polyelectrolytes were used in our studies: poly(allylamine) hydrochloride (PAH), of a cationic type, and poly(sodium 4-styrenesulfonate) (PSS) of an anionic type, both having molecular weight of 70,000. The bulk characteristics of polymers were determined by measuring the specific density, diffusion coefficient for various ionic strengths, and zeta potential. These measurements as well as molecular dynamic simulations of chain shape and configurations suggested that the molecules assume an extended, wormlike shape in the bulk. Accordingly, the diffusion coefficient was interpreted in terms of a simple hydrodynamic model pertinent to flexible rods. These data allowed a proper interpretation of polyelectrolyte multilayer adsorption from NaCl solutions of various concentrations or from 10(-3) M Tris buffer. After completing a bilayer, periodic variations in the apparent zeta potential between positive and negative values were observed for multilayers terminated by PAH and PSS, respectively. These limiting zeta potential values correlated quite well with the zeta potential of the polymers in the bulk. The stability of polyelectrolyte films against prolonged washing (reaching 26 h) also was determined using the streaming potential method. It was demonstrated that the PSS layer was considerably more resistant to washing, compared to the PAH layer. It was concluded that the experimental data were consistent with the model postulating particle-like adsorption of polyelectrolytes with little chain interpenetration. It also was concluded that due to high sensitivity, the electrokinetic method applied can be effectively used for quantitative studies of polyelectrolyte adsorption, desorption, and reconformation.