Interaction of albumins and heparinoids investigated by affinity capillary electrophoresis and free flow electrophoresis.

A fast and precise affinity capillary electrophoresis (ACE) method has been applied to investigate the interactions between two serum albumins (HSA and BSA) and heparinoids. Furthermore, different free flow electrophoresis methods were developed to separate the species which appears owing to interaction of albumins with pentosan polysulfate sodium (PPS) under different experimental conditions. For ACE experiments, the normalized mobility ratios (∆R/Rf ), which provided information about the binding strength and the overall charge of the protein-ligand complex, were used to evaluate the binding affinities. ACE experiments were performed at two different temperatures (23 and 37°C). Both BSA and HSA interact more strongly with PPS than with unfractionated and low molecular weight heparins. For PPS, the interactions can already be observed at low mg/L concentrations (3 mg/L), and saturation is already obtained at approximately 20 mg/L. Unfractionated heparin showed almost no interactions with BSA at 23°C, but weak interactions at 37°C at higher heparin concentrations. The additional signals also appeared at higher concentrations at 37°C. Nevertheless, in most cases the binding data were similar at both temperatures. Furthermore, HSA showed a characteristic splitting in two peaks especially after interacting with PPS, which is probably attributable to the formation of two species or conformational change of HSA after interacting with PPS. The free flow electrophoresis methods have confirmed and completed the ACE experiments.

Metal ion - Dehydrin interactions investigated by affinity capillary electrophoresis and computer models.

Dehydrins are specialized proteins which are related to environmental stress tolerance in plants. The proteins can bind different metal ions and have versatile other functions such as reduction of reactive oxygen species and acting as transcription factor. The structure determination of proteins from this family is challenging, since they have a high number of disordered structure elements. Consequently, to determine the functionality of these proteins on a molecular basis a computed model is helpful. This work focuses on a model for the Arabidopsis thaliana dehydrin AtHIRD11. To develop a model which reflects experimental data from literature and own binding data from affinity capillary electrophoresis experiments, a more rigid state of this protein was chosen. The Cu2+-complex of this protein was formed and evaluated. The model explains some of the properties of the complexes. Possible Cu2+-bindings site were found and the change of conformations were investigated via molecular dynamics simulation. The AtHIRD11-Cu2+-complex is a first approach towards a complex model for a structural versatile protein, which is already sufficient to explain binding data and possible structure elements.

Using affinity capillary electrophoresis and computational models for binding studies of heparinoids with p-selectin and other proteins.

A fast and precise affinity capillary electrophoresis (ACE) method has been developed and applied for the investigation of the binding interactions between P-selectin and heparinoids as potential P-selectin inhibitors in the presence and absence of calcium ions. Furthermore, model proteins and vitronectin were used to appraise the binding behavior of P-selectin. The normalized mobility ratios (∆R/Rf ), which provided information about the binding strength and the overall charge of the protein-ligand complex, were used to evaluate the binding affinities. It was found that P-selectin interacts more strongly with heparinoids in the presence of calcium ions. P-selectin was affected by heparinoids at the concentration of 3 mg/L. In addition, the results of the ACE experiments showed that among other investigated proteins, albumins and vitronectin exhibited strong interactions with heparinoids. Especially with P-selectin and vitronectin, the interaction may additionally induce conformational changes. Subsequently, computational models were applied to interpret the ACE experiments. Docking experiments explained that the binding of heparinoids on P-selectin is promoted by calcium ions. These docking models proved to be particularly well suited to investigate the interaction of charged compounds, and are therefore complementary to ACE experiments.

Ca(2+) -complex stability of GAPAGPLIVPY peptide in gas and aqueous phase, investigated by affinity capillary electrophoresis and molecular dynamics simulations and compared to mass spectrometric results.

Strong, sequence-specific gas-phase bindings between proline-rich peptides and alkaline earth metal ions in nanoESI-MS experiments were reported by Lehmann et al. (Rapid Commun. Mass Spectrom. 2006, 20, 2404-2410), however its relevance for physiological-like aqueous phase is uncertain. Therefore, the complexes should also be studied in aqueous solution and the relevance of the MS method for binding studies be evaluated. A mobility shift ACE method was used for determining the binding between the small peptide GAPAGPLIVPY and various metal ions in aqueous solution. The findings were compared to the MS results and further explained using computational methods. While the MS data showed a strong alkaline earth ion binding, the ACE results showed nonsignificant binding. The proposed vacuum state complex also decomposed during a molecular dynamic simulation in aqueous solution. This study shows that the formed stable peptide-metal ion adducts in the gas phase by ESI-MS does not imply the existence of analogous adducts in the aqueous phase. Comparing peptide-metal ion interaction under the gaseous MS and aqueous ACE conditions showed huge difference in binding behavior.

Precise small volume sample handling for capillary electrophoresis.

CE is one of the most important analytical techniques. Although the injected sample volume in CE is only in the nanoliter range, most commercial CE instruments need approximately 50 µL of the sample in the injection vial to perform the analysis. Hence, in order to fully profit from the low injection volumes, smaller vial volumes are required. Thus experiments were performed using silicone oil, which has higher density than water (1.09 g/mL) to replace sample dead volume in the vial. The results were compared to those performed without using the silicone oil in the sample vial. As an example five standard proteins namely beta-lactoglobulin, BSA, HSA, myoglobin, and ovalbumin, and one of the coagulation cascade involved proteins called vitonectin were investigated using CE. Mobility ratios and peak areas were compared. However, no significant changes were observed (RSDs% for mobility ratios and peak areas were better than 0.9 and 5.8%, respectively). Afterwards, an affinity CE method was used to investigate the interactions of two proteins, namely HSA and vitronectin, with three ligands namely enoxaparin sodium, unfractionated heparin, and pentosan polysulfate sodium. Mobility shift precision results showed that the employment of the filling has no noticeable effect on any of the protein-ligand interactions. Using a commercial PrinCE instrument and an autosampler the required sample volume is reduced down to 10 µL, and almost this complete volume can be subsequently injected during repeated experiments.