Congophilicity (Congo red affinity) of different beta2-microglobulin conformations characterized by dye affinity capillary electrophoresis

The amyloidogenic protein beta-microglobulin was characterized by affinity capillary electrophoresis (CE). CE could separate conformational variants of beta2-microglobulin and with the amyloid-specific dye Congo red as a buffer additive it was possible to measure different Congo red-affinities of native and abnormally folded beta2-microglobulin. We find that native beta2-microglobulin has an intermediate affinity for Congo red at pH 7.3 and that binding involves electrostatic interactions. The conformational variant of beta2-microglobulin that appears in acetonitrile solutions binds Congo red more strongly. Affinity CE using Congo red as a buffer additive is a new, simple, fast, and quantitative micromethod for the characterization of soluble conformational intermediates of amyloidogenic proteins.     

Sulfonated molecules that bind a partially structured species of beta2-microglobulin also influence refolding and fibrillogenesis

Human beta2-microglobulin (beta2-m) is a small amyloidogenic protein responsible for dialysis-related amyloidosis, which represents a severe complication of long-term hemodialysis. A therapeutic approach for this amyloidosis could be based on the stabilization of beta2-m through the binding to a small molecule, to possibly inhibit protein misfolding and amyloid fibril formation. The search of a strong ligand of this protein is extremely challenging: by using CE in affinity and refolding experiments we study the effect that previously selected sulfonated molecules have on the equilibrium between the native form and an ensemble of conformers populating the slow phase of beta2-m folding. These data are correlated with the effect that the same molecules exert on in vitro fibrillogenesis experiments.     

Search of ligands for the amyloidogenic protein beta2-microglobulin by capillary electrophoresis and other techniques

Beta2-microglobulin (beta2-m) is a small amyloidogenic protein normally present on the surface of most nucleated cells and responsible for dialysis-related amyloidosis, which represents a severe complication of long-term hemodialysis. A therapeutic approach for this amyloidosis could be based on the stabilization of beta2-m through the binding to a small molecule, and consequent inhibition of protein misfolding and amyloid fibril formation. A few compounds have been described to weakly bind beta2-m, including the drug suramin. The lack of a binding site for nonpolypeptidic ligands on the beta2-m structure makes it difficult for both the identification of functional groups responsible for the binding and the search of hits to be optimized. The characterization of the binding properties of suramin for beta2-m by using three different techniques (surface plasmon resonance, affinity CE (ACE), ultrafiltration) is here described and the results obtained are compared. The common features of the chemical structures of the compounds known to bind the protein led us to select 200 sulfonated/suramin-like molecules from a wider chemical library on the basis of similarity rules, so as to possibly single out some interesting hits and to gain more information on the functional groups involved in the binding. The development of screening methods to test the compounds by using ultrafiltration and ACE is described.     

Capillary electrophoresis of polycationic poly(amidoamine) dendrimers

Generation 2 to generation 5 poly(amidoamine) (PAMAM) dendrimers having different terminal functionalities were analyzed by capillary electrophoresis (CE). Polyacrylamide gel electrophoresis was also used to assess the composition of the individual generations for comparison with the CE results. Separation of PAMAMs can be accomplished by either using uncoated silica or silanized silica capillaries, although reproducibility is poor using the uncoated silica capillary. To improve run-to-run reproducibility, silanized capillary was used and various internal standards were also tested. Relative and normalized migration times of primary amine terminated PAMAM dendrimers were then determined using 2,3-diaminopyridine (2,3-DAP) as an internal standard. Using silanized capillaries and internal standards, the relative and normalized migration times are fully reproducible and comparable between runs. Apparent dimensionless electrophoretic mobilities were determined and the results were compared to theoretical calculations. It is concluded that for PAMAMs a complex separation mechanism has to be considered in CE, where the movement of the ions is due to the electric field, but the separation is rather the consequence of the adsorption/desorption equilibria on the capillary wall ("electrokinetic capillary chromatography"). The described method may be used for quality control and may serve as an effective technique to analyze polycationic PAMAM dendrimers and their derivatives with different surface modifications.     

Capillary electrophoresis of Cr(VI) reducer Arthrobacter oxydans

Rapid and effective separation of bacteria Arthrobacter oxydans was performed using capillary electrophoresis. For optimal separation of bacteria the influence of buffer concentration, pH and applied voltage were studied. It was found that the most appropriate conditions for electrophoretic mobility measurements are as follows: applied voltage 6-14 kV; buffer concentration 5-10 mM pH 6-8. At the stationary phase of growth there are always two main heterogeneous peaks. They are connected with the morphology of bacteria as well as with cell aggregation. The heterogeneity of samples may be explained by surface modifications of bacterial cells.     

Capillary electrophoresis ribosomal RNA single-stranded conformation polymorphism: a new approach for characterization of low-diversity microbial communities

Capillary electrophoresis (CE) has been the principle system for nucleic acid analysis since the early 1990s due to its inherent advantages such as fast analysis time, high resolution and efficiency, minimal sample requirement, high detection sensitivity, and automation. In the past few decades, microbial community fingerprinting methods such as terminal restriction fragment length polymorphism and single-stranded conformation polymorphism (SSCP) have migrated to CE to utilize its advantages over conventional slab gel electrophoresis. Recently, a gel-based direct rRNA fingerprint method was demonstrated. Different from other existing microbial community characterization approaches, this novel approach is polymerase chain reaction free and capable of providing information on the relative abundance of rRNA from individual phylotypes in low-diversity samples. As a gel-based method, it has a long analysis time and relatively large reagent and sample requirements. Here, we addressed these limitations by transferring the RNA fingerprint approach to the CE platform. Analysis time significantly improved from 24?h to 60?min, and the use of a fluorescently labeled hybridization probe as the detection strategy decreased the sample requirement by ten-fold. The combination of fast analysis time, low sample requirement, and sensitive fluorescence detection makes CE-RNA-SSCP an appealing new approach for characterizing low-diversity microbial communities.     

Chain microstructure and conformation of partially quaternized poly(dimethylaminoethyl methacrylate)

¹H-NMR spectroscopy was used to establish the chain microstructure and conformation produced by the quaternization reaction of syndiotactic poly[2-(dimethylamino)ethyl methacrylate], a polymer with tertiary amino groups in the side chains. A chain microstructure with mini blocks of modified units was found by analyzing the N⁺CH₃ signal that was proved to be split in accordance with the composition triads. The macromolecular backbone changes its form by quaternization, from a close to an expanded coil, was confirmed by light scattering measurements and NOE spectra modifications. The two linked processes, the block formation and chain expansion can be the key in developing a reaction mechanism explaining both positive and negative deviations from a second-order kinetic model.     

The influence of Cu(2+) on the unfolding and refolding of intact and proteolytically processed beta(2)-microglobulin

Human beta(2)-microglobulin (beta(2)m) is an amyloidogenic protein in patients suffering from chronic kidney disease and especially in those patients that need intermittent hemodialysis for longer periods, e.g., when awaiting transplantation. While many in vitro conditions induce beta(2)m-amyloid formation from wild-type (wt) beta(2)m and while a number of structurally altered beta(2)m molecules are known to be conformationally unstable and amyloidogenic on their own, it is not known why beta(2)m-amyloid is generated in some dialysis patients. For many amyloid proteins it is known that divalent metal ions, especially Cu(2+), display strong binding and distinct destabilizing effects on protein conformation. The present study uses CE to assess conformational states of wt and cleaved beta(2)m (dK58-beta(2)m, beta(2)m cleaved at lysine-58, a modification found in the circulation of hemodialysis patients) in the presence of divalent metal ions. The experiments provide both qualitative and quantitative data showing the specific destabilizing effects of Cu(2+)-ions on the folding of wt beta(2)m. Both refolding after acid denaturation and solution structure of beta(2)m under otherwise native conditions are severely influenced by Cu(2+). An increased unfolding, aggregation, and induction of Congo red-reactive molecular species in Cu(2+)-incubated wt-beta(2)m could be demonstrated while the refolding kinetics of dK58-beta(2)m, already slower than the wt molecule, appeared not to be further decreased by Cu(2+). Given the interest in the actions of metal ions in other types of amyloidosis, including, e.g., Alzheimer's disease and the prion encephalopathies, the use of microelectrophoretic methods to monitor unfolding and refolding of biomolecules available in scarce amounts as shown in this study is an attractive option.     

Capillary zone electrophoresis of amino acids on a hybrid poly(dimethylsiloxane)-glass chip

Poly(dimethylsiloxane) (PDMS)-PDMS and hybrid PDMS-glass devices have been characterized and compared in terms of current-voltage linearity, contact angle, electroosmotic velocity, electroosmotic mobility, and electrokinetic potential in dependence on the surface treatment. The hybrid PDMS-glass microfluidic devices have further been tested as on-chip capillary electrophoresis systems for the separation of fluorescently labeled amino acids. It has been demonstrated that different methods of surface pretreatment of the PDMS-glass devices result in significantly different separation performance, with plate numbers varying from 650 to 57 000 in dependence on the surface state and the nature of the amino acids. Electrophoretic separations of amino acids have been achieved within tens of seconds with detection limits of less than 2 microM (approximately 2 x 10(-16) to 2.5 x 10(-16) mol quantities at injection volumes of 110-120 pL). The detected amounts of fluorescein isothiocyante (FITC)-amino acids are at least ten times lower, since the amino acid:FITC ratio is 10:1 mol. The results demonstrate the perspective of such hybrid PDMS-glass microfluidic systems and the methods to modify their surfaces for on-chip separation methods for biomolecules.     

Capillary zone electrophoresis investigation of the interaction between heparin and granulocyte-colony stimulating factor

The interaction between standard heparin, low-molecular-weight heparin (LMWH), and granulocyte-colony stimulating factor (G-CSF) was studied by capillary zone electrophoresis. Both qualitative and quantitative characterizations of the heparin-protein binding were determined. The binding constants of the two different groups of heparins with G-CSF, calculated from the Scatchard plot by regression, were 4.805 x 10(5) M(-1) and 4.579 x 10(5) M(-1), respectively. The two binding constants measured are of the same order of magnitude at 10(5) M(-1), indicating that LMWH contains most of the functional groups bound to G-CSF by standard heparin.     

Capillary zone electrophoresis of proteins with poly(2-hydroxyethyl methacrylate)-coated capillaries: fundamentals and applications

Fused silica capillaries have been modified by atom-transfer radical polymerization (ATRP) to generate covalently bonded polymer films of 2-hydroxyethyl methacrylate. Because the kinetics of ATRP have mainly been investigated in bulk solutions, a GC experiment was set up to examine monomer conversion inside narrow-bore capillaries. It was shown that after 1 to 4 h the reaction was nearly complete. The coating process was further optimized by monitoring EOF, because low EOF indicates high surface coverage. To deal with the very low EOF values, a new approach was used to dramatically reduce the measurement time by overlaying hydrodynamic flow on the electroosmotic flow. The corresponding equations are derived separately in detail. Capillaries were then coated under optimum conditions with linear or cross-linked polymer films. The EOF was reduced over a wide range of pH values. A long-term reproducibility test with both types of functionalization showed that the efficiency of the linear polymer coating decreased significantly over time. With cross-linked films, however, the efficiency even increased. Relative standard deviations for protein migration times were also much lower in cross-linked coated capillaries. Highly efficient separations could be performed for basic and acidic proteins in acidic media, and for the latter even in basic media. Received: 14 September 2000 / Revised: 26 November 2000 / Accepted: 30 November 2000     

Capillary zone electrophoresis of proteins with poly(2-hydroxyethyl methacrylate)-coated capillaries: fundamental and applications

Fused silica capillaries have been modified by atom-transfer radical polymerization (ATRP) to generate covalently bonded polymer films of 2-hydroxyethyl methacrylate. Because the kinetics of ATRP have mainly been investigated in bulk solutions, a GC experiment was set up to examine monomer conversion inside narrow-bore capillaries. It was shown that after 1 to 4 h the reaction was nearly complete. The coating process was further optimized by monitoring EOF, because low EOF indicates high surface coverage. To deal with the very low EOF values, a new approach was used to dramatically reduce the measurement time by overlaying hydrodynamic flow on the electroosmotic flow. The corresponding equations are derived separately in detail. Capillaries were then coated under optimum conditions with linear or cross-linked polymer films. The EOF was reduced over a wide range of pH values. A long-term reproducibility test with both types of functionalization showed that the efficiency of the linear polymer coating decreased significantly over time. With cross-linked films, however, the efficiency even increased. Relative standard deviations for protein migration times were also much lower in cross-linked coated capillaries. Highly efficient separations could be performed for basic and acidic proteins in acidic media, and for the latter even in basic media.     

Capillary electrophoresis investigation on the structure-enantioselectivity relationship in synthetic cyclopeptides as chiral selectors

We recently reported the use of a deconvolution strategy to identify the best chiral selectors for Nalpha-2,4-dinitrophenyl (Dnp) amino acid racemates from a combinatorial library composed of thousands of homodetic cyclohexapeptides. Selection was based on the capillary electrophoresis (CE) enantioresolution for a set of Dnp-amino acids. The groups involved in the chiral discrimination were assessed by nuclear magnetic resonance (NMR) spectroscopy, which revealed a strong involvement of one of the aromatic rings of the cyclopeptide in the binding with the analyte. In order to better understand the recognition mechanism, and thus extend the applicability of the analytical system, modifications on both analyte and selector structure were introduced. The effects on separation were evaluated in terms of resolution values and mobility variation.