The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins

Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized onto immobilized artificial membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC (U87MG) column, and the binding affinities (K_d) determined were 1.08 ± 0.49 and 0.0086 ± 0.0006 μM, respectively, consistent with previously reported values. Furthermore, binding affinities (K_i) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX, and rotenone. In addition, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC (U87MG) was consistent with the obtained K_i values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy.     

Design of salt‐tolerant membrane adsorbers for viral clearance

Strong anion exchange chromatography has frequently been employed as a viral clearance step during downstream processing of biological therapeutics. When challenged with viruses having only slightly acidic isoelectric points, the performance of the anion exchange operation becomes highly dependent on the buffer salt concentration, with the virus log reduction value (LRV) dropping dramatically in buffers with 50–150 mM salt. In this work, a series of anion exchange membrane adsorbers utilizing alternative ligand chemistries instead of the traditional quaternary amine (Q) ligand have been developed that overcome this limitation. Four different ligands (agmatine, tris‐2‐aminoethyl amine, polyhexamethylene biguanide, and polyethyleneimine) achieved >5 LRV of bacteriophage ΦX174 (pI ～ 6.7) at pH 7.5 and up to 150 mM salt, compared to 0 LRV for the Q ligand. By evaluating structural derivatives of the successful ligands, three factors were identified that contributed to ligand salt tolerance: ligand net charge, ligand immobilization density on the membrane, and molecular structure of the ligand‐binding group. Based on the results of this study, membrane adsorbers that incorporate alternative ligands provide a more robust and salt tolerant viral clearance‐processing step compared to traditional strong anion exchange membrane adsorbers. Biotechnol. Bioeng. 2009;103: 920–929. © 2009 Wiley Periodicals, Inc.     

Affinity purification of proteins using ligands derived from peptide libraries

Peptide libraries can be used to identify ligands that bind specifically to a desired protein. These peptides may have significant advantages as specific ligands for affinity chromatography separations. This article describes the use of one of such peptide, Try-Asn-Phe-Glu-Val-Leu, as a ligand for the purification of S-protein using affinity chromatography. General strategies for peptide immobilization are discussed and the conditions for peptide immobilization to Emphazetrade mark gel are optimized. The effects of peptide orientation and peptide densities on protein binding are studied. Results indicate that the peptide affinity is not affected by the orientation of the peptide during immobilization, but association constants can be reduced by one order of magnitude when compared with the values in solution.With increased peptide density, the protein binding capacity of the gel increases, but both the percentage of peptide utilization and apparent binding constant between immobilized peptide and S-protein decrease. S-protein is separated from a mixture with BSA via affinity chromatography using specific elution with the peptide in solution.Finally, direct purification of S-protein from an enzymatic digestion mixture of ribonuclease A is demonstrated.(c) 1995 John Wiley & Sons, Inc.     

Drug affinity to immobilized target bio-polymers by high-performance liquid chromatography and capillary electrophoresis

This review addresses the use of high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) as affinity separation methods to characterise drugs or potential drugs-bio-polymer interactions. Targets for the development of new drugs such as enzymes (IMERs), receptors, and membrane proteins were immobilized on solid supports. After the insertion in the HPLC system, these immobilized bio-polymers were used for the determination of binding constants of specific ligands, substrates and inhibitors of pharmaceutical interest, by frontal analyses and zonal elution methods. The most used bio-polymer immobilization techniques and methods for assessing the amount of active immobilized protein are reported. Examples of increased stability of immobilized enzymes with reduced amount of used protein were shown and the advantages in terms of recovery for reuse, reproducibility and on-line high-throughput screening for potential ligands are evidenced. Dealing with the acquisition of relevant pharmacokinetic data, examples concerning human serum albumin binding studies are reviewed. In particular, papers are reported in which the serum carrier has been studied to monitor the enantioselective binding of chiral drugs and the mutual interaction between co-administered drugs by CE and HPLC. Finally CE, as merging techniques with very promising and interesting application of microscale analysis of drugs' binding parameters to immobilized bio-polymers is examined.     

Development of a homogeneous high throughput fluorescence polarization assay for G protein-coupled receptor binding

Traditional methods that follow receptor ligand interactions are competitive assays in which the test compound displaces a radiolabeled molecule. These assays require either a time-consuming step for separation of free ligands from bound ligands or immobilization of receptors and the scintillant on a solid-phase support. In this report, we describe the development of a homogeneous binding assay for a G protein-coupled receptor in the fluorescence polarization format. This homogeneous fluorescence polarization binding assay format is superior to the traditional binding methods because no radioisotope, separation step, or solid-phase support is required. The elimination of the separation step enhances detection of low-affinity ligands and enables a real-time, continuous readout of the binding activity in a high throughput 384-well microplate format.     

Adsorptive immobilization of erythrocyte membrane

Immobilization of human erythrocyte membrane was carried out by adsorption on Fractosil, a porous form of silica. Acetylcholinesterase (AChE) was chosen as a representative membrane enzyme in this study. Dependency of adsorption on membrane concentration was determined. Positive cooperative interactions that occurred in the process of immobilization increased stability. Presence of hydrophobic ligands on derivatized Fractosil was found to enhance stability of immobilized preparations making them more effective for use in continuous catalytic transformations. It is suggested that adsorptive immobilization of membrane structures such as the human erythrocyte membrane fragments on Fractosil and other inexpensive supports may provide a convenient procedure for utilization of their catalytic potential. Such preparations may be used in diagnostic kits or for construction of biosensors.     

Facile construction of fluorescent peptide microarrays: One-step fluorescent derivatization of sub-microscale peptide aldehydes for selective terminal immobilization

In this note, we demonstrate the utility of bifunctional fluorescent linkers to facilitate the construction of peptide microarrays with either an N- or a C-terminal alkylamine for directionally preferred peptide immobilization. Significantly, these small tags facilitate high-performance liquid chromatography (HPLC) profiling while limiting interference with antigen-antibody interactions after peptide immobilization. In a model peptide-antibody binding assay, a sequence-dependent orientation effect of antibody binding to a series of peptide ligands was demonstrated. This approach provides a strategy that can be applied to a variety of peptide microarray-based detection systems.     

Selective immobilization of multivalent ligands for surface plasmon resonance and fluorescence microscopy

Cell surface multivalent ligands, such as proteoglycans and mucins, are often tethered by a single attachment point. In vitro, however, it is difficult to immobilize multivalent ligands at single sites due to their heterogeneity. Moreover, multivalent ligands often lack a single group with reactivity orthogonal to other functionality in the ligand. Biophysical analyses of multivalent ligand-receptor interactions would benefit from the availability of strategies for uniform immobilization of multivalent ligands. To this end, we report the design and synthesis of a multivalent ligand that has a single terminal orthogonal functional group and we demonstrate that this material can be selectively immobilized onto a surface suitable for surface plasmon resonance (SPR) experiments. The polymeric ligand we generated displays multiple copies of 3,6-disulfogalactose, and it can bind to the cell adhesion molecules P- and L-selectin. Using SPR measurements, we found that surfaces displaying our multivalent ligands bind specifically to P- and L-selectin. The affinities of P- and L-selectin for surfaces displaying the multivalent ligand are five- to sixfold better than the affinities for a surface modified with the corresponding monovalent ligand. In addition to binding soluble proteins, surfaces bearing immobilized polymers bound to cells displaying L-selectin. Cell binding was confirmed by visualizing adherent cells by fluorescence microscopy. Together, our results indicate that synthetic surfaces can be created by selective immobilization of multivalent ligands and that these surfaces are capable of binding soluble and cell-surface-associated receptors with high affinity.     

Selective adsorption of endotoxin inside a polycationic network of flat-sheet microfiltration membranes

For the removal of remaining amounts of endotoxin, sorbents with high selectivity for endotoxin are required. Typically, particulate sorbents with positively charged ligands, such as histidine, polymyxin B poly-L-lysine and poly(ethyleneimine) (PEI), display moderate to high removal efficiencies in an environment of low ionic strength. It was found that polycationic ligands are most suitable to meet an endotoxin concentration which is below the threshold level required for parenteralia. Furthermore, protein recoveries close to 100% are obtained if the decontamination is performed at a pH close to the pI of acidic proteins. The high selectivity is probably caused by complexation of the polycationic ligand with the polyanionic endotoxin, leading to interactions with KD < 10(-9) M using PEI and assuming M(r) = 10 kDa for monomeric endotoxin; with BSA the same ligand reveals only KD = 4 x 10(-6) M. Using polymer-coated microfiltration membranes, immobilization of positively charged ligands leads to membrane adsorbers which are generally superior to chromatographic adsorbers and allow faster processing. Since immobilization takes place at polymer chains, low-molecular-weight ligands mainly add positive charges to the hydrophilic polymer. Consequently, membrane adsorbers with low-molecular-weight ligands, even DEAE, demonstrate similar selectivity to PEI or poly-L-lysine.     

Binding properties of antimicrobial agents to dipeptide terminal of lipid II using surface plasmon resonance

We developed a surface plasmon resonance (SPR) assay to estimate the interactions of antimicrobial agents with the dipeptide terminal of lipid II (d-alanyl-d-alanine) and its analogous dipeptides (l-alanyl-l-alanine and d-alanyl-d-lactate) as ligands. The established SPR method showed the reproducible immobilization of ligands on sensor chip and analysis of binding kinetics of antimicrobial agents to ligands. The ligand-immobilized chip could be used repeatedly for at least 200 times for the binding assay of antimicrobial agents, indicating that the ligand-immobilized chip is sufficiently robust for the analysis of binding kinetics. In this SPR system, the selective and specific binding characteristics of vancomycin and its analogs to the ligands were estimated and the kinetic parameters were calculated. The kinetic parameters revealed that one of the remarkable binding characteristics was the specific interaction of vancomycin to only the d-alanyl-d-alanine ligand. In addition, the kinetic binding data of SPR showed close correlation with the antimicrobial activity. The SPR data of other antimicrobial agents (e.g., teicoplanin) to the ligands showed correlation with the antimicrobial activity on the basis of the therapeutic mechanism. Our SPR method could be a valuable tool for predicting the binding characteristics of antimicrobial agents to the dipeptide terminal of lipid II.     

Cellulose and glass fiber affinity membranes for the chromatographic separation of biomolecules

Macroporous cellulose and glass membranes were prepared from filter paper and glass fiber filter, respectively. To enhance their stability, the cellulose membranes were crosslinked with epichlorohydrin, and the glass membranes were crosslinked with glutaraldehyde or organic bifunctional silanes. Several pathways for the modification, activation, and ligand immobilization were used and compared. For cellulose membranes, the diazotization method provided the best results, whereas the glutaraldehyde method provided the best performance for glass membranes, regarding both their stability and ligand immobilization capacity. The characterization of the membranes was made by using a triazine dye, bovine serum albumin, and trypsin as test ligands. The membrane morphologies and the uniformities of ligand distribution across the membrane cartridges were investigated. Numerous affinity ligands were immobilized onto the membranes, and the prepared affinity membranes have been used to separate or purify concanavalin A, peroxidase, protease inhibitors, globulin, fibronectin, and other biomolecules.     

Affibody protein capture microarrays: synthesis and evaluation of random and directed immobilization of affibody molecules

Affibody molecules, 58-amino acid three-helix bundle proteins directed to different targets by combinatorial engineering of staphylococcal protein A, were used as capture ligands on protein microarrays. An evaluation of slide types and immobilization strategies was performed to find suitable conditions for microarray production. Two affibody molecules, Z(Taq) and Z(IgA), binding Taq DNA polymerase and human IgA, respectively, were synthesized by solid phase peptide synthesis using an orthogonal protection scheme, allowing incorporation of selective immobilization handles. The resulting affibody variants were used for random surface immobilization (through amino groups) or oriented surface immobilization (through cysteine or biotin coupled to the side chain of Lys58). Evaluation of the immobilization techniques was carried out using both a real-time surface plasmon resonance biosensor system and a microarray system using fluorescent detection of Cy3-labeled target protein. The results from the biosensor analyses showed that directed immobilization strategies significantly improved the specific binding activity of affibody molecules. However, in the microarray system, random immobilization onto carboxymethyl dextran slides and oriented immobilization onto thiol dextran slides resulted in equally good signal intensities, whereas biotin-mediated immobilization onto streptavidin-coated slides produced slides with lower signal intensities and higher background staining. For the best slides, the limit of detection was 3 pM for IgA and 30 pM for Taq DNA polymerase.     

Influence of azide incorporation on binding affinity by small papain inhibitors

In order to develop affinity-based biosensor platforms, appropriate ligands with a functional handle for immobilization onto a biosensor surface are required. To this end, a library of papain inhibitors was designed and synthesized, containing different azide linkers for subsequent immobilization by ‘click’ chemistry, in this particular case by copper-free, strain-promoted azide–alkyne cycloaddition (SPAAC). Furthermore, a molecular docking study was performed to obtain a better insight as to at which position such azide handles could be tolerated without affecting binding affinity. Although the azide moiety is small, in some cases its introduction strongly influenced the binding affinity. For one class of inhibitors a swapped binding mode was proposed to explain the results. In addition, a specific site for linker introduction was identified, which did not significantly affect the binding affinity.     

Biochemical studies on muscarinic receptors

Muscarinic receptors have been characterized in smooth muscle and brain by the binding of reversible (e.g. atropine, quinuclidinylbenzylate) or irreversible (benzilylcholine or propylbenzilylcholine mustards) ligands. There is a close correlation between affinity constants derived from binding experiments and the affinities of muscarinic ligands for these sites obtained in pharmacological experiments on smooth muscle. Whereas atropine shows a single high affinity binding component (in subcellular preparations) several other ligands (QNB, ACh, oxotremorine) show multiple affinity binding. This indicated the existence of several types of binding sides which show selectivity toward certain cholinergic effectors. Most detergents inhibit the binding of ligands to the receptor site and therefore cannot be used to solubilize the receptor protein from the membrane. Treatment of brain subcellular membrane preparations with high salt concentrations (2M NaI) solubilize proteins which possess the muscarinic ligand binding properties observed in the membrane preparation. The affinities for muscarinic antagonists however are decreased, which suggests that a conformational change occurs in the protein upon solubilization.     

Cooperative action between band 3 and glycophorin A in human erythrocytes: immobilization of band 3 induced by antibodies to glycophorin A.

The ability of transmembrane receptor proteins to change their association with the cytoskeleton in response to ligand binding seems to be a key mechanism of signal transduction across membranes. To investigate the molecular features of this mechanism we have used the red cell membrane as a model system to study signal transduction through the integral protein, glycophorin A. In these studies the lateral mobility of integral proteins was measured in situ by fluorescence recovery after photobleaching, and membrane rigidity was characterized by micropipette aspiration technique. We found that binding either a monoclonal antibody or its monovalent Fab to the exoplasmic domain of glycophorin A in normal red cells immobilized the receptor and rigidified the membrane. Further, immobilization and rigidification did not occur when antibodies were bound to Miltenberger V cells containing a mutant form of glycophorin A lacking the cytoplasmic domain. These results imply that the site of the immobilization/rigidification lies within the membrane skeletal structure, not in exofacial receptor crosslinking, and requires the extended cytoplasmic domain of normal glycophorin A. In addition, we found that glycophorin A immobilization and membrane skeletal rigidification were accompanied by immobilization of band 3 receptors. This unexpected result indicates a cooperative coupling between liganded glycophorin A, band 3, and the membrane skeleton. We speculate that cooperation of this type may represent a general mechanism for cytoskeletal linkage and transformation initiated by receptors with short cytoplasmic sequences, such as integrins.     

Purification of soluble and membrane-bound proteases with substrate-analogous inhibitors by affinity chromatography.

Specific modified substrate-analogous amino acids and peptides have been used as affinity ligands in the affinity chromatography of proteases. Alanine methyl ketone-Sepharose (AMK-Sepharose) is introduced as affinity support for the purification of a bacterial alanyl aminopeptidase (AAP) from a membrane protein extract and Arginine-Agarose as support for the preparation of a membrane-bound proteinase of myeloma cells (MP-1). Peptidyl methyl ketones as affinity ligands have been used to separate subtilisin enzymes and the cysteine proteases cathepsin B, L, and S. As a new type of ligands, spacer-bound peptidyl chloromethyl ketones are presented for a specific and oriented immobilization of proteinases. Oriented-immobilized cathepsin B was used to isolate antibodies against this enzyme.     

Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules is reported. The entire sequence of relevant experimental steps is described, involving the fabrication of nanostructured substrates using electron beam lithography, immobilization of biomolecules on the substrates, and their characterization utilizing surface-enhanced Raman spectroscopy (SERS). Three different designs of nano-biological systems are employed, including protein A, glucose binding protein, and a dopamine binding DNA aptamer. In the latter two cases, the binding of respective ligands, D-glucose and dopamine, is also included. The three kinds of biomolecules are immobilized on nanostructured substrates by different methods, and the results of SERS imaging are reported. The capabilities of SERS to detect vibrational modes from surface-immobilized proteins, as well as to capture the protein-ligand and aptamer-ligand binding are demonstrated. The results also illustrate the influence of the surface nanostructure geometry, biomolecules immobilization strategy, Raman activity of the molecules and presence or absence of the ligand binding on the SERS spectra acquired.     

Calcium alginate immobilization of mammalian neurons: A potential tool to purify ligands of neuronal membrane proteins

Summary We report here improved immobilization conditions which permitted (i) to immobilize mouse neuroblastoma cells in calcium alginate beads, (ii) to test the functions of using patch clamp techniques and (iii) to quantitatively analyze ligand interactions with voltage-dependent sodium channels in neurons immobilized inside alginate beads. These results qualify this immobilization technique as a biotechnological tool to isolate and/or purify ligands of neuronal membrane proteins. A part of these results was presented at the International Symposium “Physiology of immobilized Cells” at Wageningnen, The Netherlands, December 10–13, 1989     

Immobilized GABAA receptors and their ligand binding characteristics

ConA-sepharose and polylysine-agarose beads effectively bound detergent-solubilized GABAA receptors from rat cerebrocortical membranes. The immobilized receptors showed a single class of high affinity binding sites specific for flunitrazepam or muscimol and displayed GABA-stimulated flunitrazepam binding. Maximal binding capacities of the ConA-immobilized receptor for the ligands were about three times greater than those of the polylysine-immobilized receptors. The relative affinities for each of the ligands were not affected by the method of receptor immobilization. The dissociation constants for muscimol of these immobilized receptors were somewhat dependent on the solubilizing agents used, but were considerably lower than those measured using extensively dialyzed rat cerebrocortical membranes.     

A ligand-receptor binding assay by receptor immobilization

Using transferrin-transferrin receptor binding as a model of ligand-receptor binding, we have developed a new and simple binding assay for the solubilized receptor. Solubilized membrane proteins containing transferrin receptor were immobilized by covalent binding to beads having chemical reactive epoxide groups, and then 125I-labeled transferrin was added to the beads. Dose-dependent, ligand-specific, and saturable binding of 125I-labeled transferrin to the immobilized membrane proteins were demonstrated and a Scatchard analysis derived affinity of Kd = 1.8 X 10(-9) M was obtained. These results indicate that the immobilization of receptors onto beads may be useful in a simple binding assay of the solubilized receptor.