A new highly adaptable design of shear-flow device for orientation of macromolecules for Linear Dichroism (LD) measurement.

This article presents a new design of flow-orientation device for the study of bio-macromolecules, including DNA and protein complexes, as well as aggregates such as amyloid fibrils and liposome membranes, using Linear Dichroism (LD) spectroscopy. The design provides a number of technical advantages that should make the device inexpensive to manufacture, easier to use and more reliable than existing techniques. The degree of orientation achieved is of the same order of magnitude as that of the commonly used concentric cylinders Couette flow cell, however, since the device exploits a set of flat strain-free quartz plates, a number of problems associated with refraction and birefringence of light are eliminated, increasing the sensitivity and accuracy of measurement. The device provides similar shear rates to those of the Couette cell but is superior in that the shear rate is constant across the gap. Other major advantages of the design is the possibility to change parts and vary sample volume and path length easily and at a low cost.

Quantitative enhanced Raman scattering of labeled DNA from gold and silver nanoparticles.

Surface-enhanced resonance Raman scattering (SERRS) from silver nanoparticles using 514.5-nm excitation has been shown to offer huge potential for applications in highly sensitive multiplexed DNA assays. If the technique is to be applied to real biological samples and integrated with other methods, then the use of gold nanoparticles and longer wavelengths of excitation are desirable. The data presented here demonstrate that dye-labeled oligonucleotide sequences can be directly detected by SERRS using gold nanoparticles in a quantitative manner for the first time. The performance of gold and silver nanoparticles as SERRS substrates was assessed using 514.5-, 632.8-, and 785-nm excitation and a range of 13 commercially available dye-labeled oligonucleotides. The quantitative response allowed the limit of detection to be determined for each case and demonstrates that the technique is highly effective, sensitive, and versatile. The possibility of excitation at multiple wavelengths further enhances the multiplexing potential of the technique. The importance of effectively combining the optical properties of the nanoparticle and the dye label is demonstrated. For example, at 632.8-nm excitation, the dye BODIPY TR-X and gold nanoparticles make a strong SERRS combination with very little background fluorescence. This study allows the choice of nanoparticle and dye label for particular experimental setups, and significantly expands the applicability of enhanced Raman scattering for use in many disciplines.

Practical control of SERRS enhancement.

The demonstration that quantitative and sensitive analysis can be carried out using surface enhanced resonance Raman scattering (SERRS) prompted a discussion and investigation of the main variables which are within the control of the analyst using colloidal silver as the substrate. Previous papers have dealt with the crucial need to obtain good chemisorption of the analyte to the surface and have reported the use of specially designed dyes for SERRS. One of the most variable processes is the aggregation of the colloid. Here, we investigate the addition of controlled amounts of an organic aggregating agent, poly-L-lysine, at concentrations which reduce the zeta potential in a controlled manner, thus aiding aggregation control. The relationship between the excitation frequency, the surface plasmon resonance frequency of the silver colloid and the frequency of the maximum absorbance of the molecular chromophore is studied using low concentrations of dye and no aggregating agent. Under these conditions, little to no aggregation is expected. The magnitude of the enhancement is strongly dependent on the frequency of the molecular chromophore as well as the plasmon resonance frequency. However, when sodium chloride is used to aggregate the colloid, a larger enhancement is obtained and the strong dependence on the molecular chromophore largely disappears. A much broader enhancement profile is obtained which appears to be related more to the specific enhancement processes caused by aggregation than the frequency of the chromophore. However, the total enhancement for SERRS is higher than for SERS thus indicating that the chromophore is still important to the process.

Effects of cholesterol and model transmembrane proteins on drug partitioning into lipid bilayers as analysed by immobilized-liposome chromatography.

We have analysed how cholesterol and transmembrane proteins in phospholipid bilayers modulate drug partitioning into the bilayers. For this purpose we determined the chromatographic retention of drugs on liposomes or proteoliposomes entrapped in gel beads. The drug retention per phospholipid amount (the capacity factor Ks) reflects the drug partitioning. Cholesterol in the bilayers decreased the Ks value and hence the partitioning into the membrane in proportion to the cholesterol fraction. On average this cholesterol effect decreased with increasing temperature. Model transmembrane proteins, the glucose transporter GLUT1 and bacteriorhodopsin, interacted electrostatically with charged drugs to increase or decrease the drug partitioning into the bilayers. Bacteriorhodopsin proteoliposomes containing cholesterol combined the effects of the protein and the cholesterol and approached the partitioning properties of red blood cell membranes. For positively charged drugs the correlation between calculated intestinal permeability and log Ks was fair for both liposomes and bacteriorhodopsin-cholesterol proteoliposomes. Detailed modeling of solute partitioning into biological membranes may require an extensive knowledge of their structures.

Advantages of quantitative affinity chromatography for the analysis of solute interaction with membrane proteins.

The use of membrane proteins as chromatographic stationary phases for the quantitation of biospecific interaction between the proteins and solutes is reviewed. This method is one among the few where a membrane protein is immobilized for repeated analyses of solute binding. To our knowledge, five transmembrane proteins have been immobilized in chromatographic matrices: the glucose and nucleoside transporters from human red blood cells, the photosynthetic reaction center from Rhodobacter sphaeroides, the nicotinic acetylcholine receptor from rat brain and a recombinant P-glycoprotein. Proteoliposomes and membrane vesicles have thereby been entrapped in size-exclusion beads, such as Superdex 200, and membrane proteins have been adsorbed on 'immobilized artificial membrane' monolayers of lipid analogs grafted to silica beads. Encouragingly, immobilized glucose transporter and P-glycoprotein showed constant interactant affinities for months. Analysis is done in the frontal mode at equilibrium because there is no separation between bound and free ligand. Both the affinity constant, which generally coincides with the corresponding constant determined by use of nonchromatographic methods, and the amount of active binding sites are obtained. The method has been successfully applied to functional analysis of membrane proteins in cells or reconstituted in lipid mono- or bilayers, screening of low-molecular interactants, investigation of protein-protein interaction and studies of effects of physico-chemical parameters on solute-protein interaction. The analyses require sensitive detection of the analyte and matching between amount of binding sites and affinity.

Chromatographic retention of drug molecules on immobilised liposomes prepared from egg phospholipids and from chemically pure phospholipids.

The partitioning of a chemically diverse set of drugs into liposomes was studied by immobilised liposome chromatography (ILC). For this purpose liposomes composed of (i) purified egg phospholipids (EPL), (ii) synthetic phosphatidylcholine (PC), (iii) PC--synthetic phosphatidylethanolamine (PE) 80:20 (mol/mol) and (iv) PC--synthetic phosphatidylserine (PS) 80:20 (mol/mol) were immobilised in gel beads by freeze-thawing. The drug partitioning was assessed from the retention volume, which was expressed as a capacity factor, K(s), normalised with respect to the amount of immobilised phospholipid. The drug retention on EPL, PC and PC--PE liposomes was very similar, whereas the negatively charged PC--PS liposomes increased the retention of positively charged and decreased retention of negatively charged drugs. The partitioning of drugs on liposome columns (log K(s)) versus their octanol--water partitioning (log P(oct)) showed three separate rectilinear relationships, depending on the charge of the compound (neutral, positive, or negative). Statistical analysis (ANCOVA) proved that the lines had similar slopes. Repeated analysis of four reference compounds showed a low variation (<0.12 log units) over time (about 250 days). A close relationship was observed between the drug retention in short EPL columns with a low content of phospholipids and the retention in longer standard EPL columns. The short 'quick screen bilayer columns' permit analysis of highly lipophilic compounds within 30 min and are thus applicable for medium-throughput screening in drug discovery settings. A very strong rectilinear relationship (r(2)=0.95, n=13) between log K(s) (EPL) and published liposome partitioning data (log D(mem)) confirmed that the ILC drug retention reflects the drug partitioning into the lipid bilayers. A moderate to fair rectilinear relationship was observed between the normalised retention on PC, PC-PE and EPL liposomes (r(2)=0.79, 0.86 and 0.85, respectively, n=24) and corresponding published log k'(IAM) data obtained on immobilised artificial membrane (IAM) columns. Transport across Caco-2 cell monolayers (log P(c)) showed curvilinear relationships with log K(s), log k'(IAM), log P(oct) and log D(oct). The drug fraction absorbed in humans showed a similar relationship to log K(s) values as to surface plasmon resonance signals representing drug-liposome interaction (Danelian et al., 2000 J Med Chem, 43, 2083--2086).

Conversion between two cytochalasin B-binding states of the human GLUT1 glucose transporter.

Two cytochalasin B-binding states of the human red blood cell facilitative glucose transporter GLUT1 were studied, one exhibiting one cytochalasin B-binding site on every second GLUT1 monomer (state 1) and the other showing one site per monomer (state 2). Quantitative affinity chromatography of cytochalasin B was performed on (a) biotinylated red blood cells, (b) cytoskeleton-depleted red blood cell membrane vesicles, and (c) GLUT1 proteoliposomes. The cells were adsorbed on streptavidin-derivatized gel beads, and the vesicles and proteoliposomes entrapped in dextran-grafted agarose gel beads. Cytochalasin B binding to free vesicles and proteoliposomes was analyzed by Hummel and Dreyer size-exclusion chromatography and ultracentrifugation. Analysis of the biotinylated cells indicated an equilibrium between the two GLUT1 states. GLUT1 in free membrane vesicles attained state 2, but was converted into state 1 on entrapment of the vesicles. Purification of GLUT1 in the presence of non-ionic detergent followed by reconstitution produced GLUT1 in state 1. This state was maintained after entrapment of the proteoliposomes. Finally, GLUT1 showed slightly higher affinity for cytochalasin B in state 1 than in state 2. In summary, the cytochalasin B-binding state of GLUT1 seemed to be affected by (a) biotinylation of the cell surface, (b) removal of the cytoskeleton at high pH and low ionic strength, (c) interaction between the dextran-grafted agarose gel matrix and the membrane vesicles, and (d) reconstitution to form proteoliposomes.

Quantitative affinity chromatographic studies of mitochondrial cytochrome c binding to bacterial photosynthetic reaction center, reconstituted in liposome membranes and immobilized by detergent dialysis and avidin--biotin binding.

In order to study the affinity binding of c-type cytochromes to the photosynthetic reaction center (RC) by quantitative affinity chromatography (QAC), RC from Rhodobacter sphaeroides was reconstituted into liposomes composed of egg phosphatidylcholine (EPC) and 2 mol% of biotinyl phosphatidylethanolamine simultaneously as the liposomes were formed and immobilized in (strept)avidin-coupled gel beads by rotary detergent dialysis. The immobilized amount was up to 80 nmol of RC and 33 micromol of lipid/g of moist gel in streptavidin-coupled Sephacryl S-1000 gel. By QAC frontal runs, retardation of mitochondrial cyt c on immobilized RC liposome columns was demonstrated. The dissociation constant for the RC-cyt c interaction was determined to be 0.20-0.57 microM. QAC studies also allowed evaluation of the orientation of reconstituted RC in immobilized liposomes by comparison of the total amount of cyt c binding sites with the amount of available binding sites obtained by QAC. It seems that the RC proteoliposomes immobilized in Sephacryl S-1000 gel exposed the cyt c binding sites on the outer surface of the liposomes due to effects of the gel network pore size and the resulting liposomal size.

Chromatography on cells: analyses of solute interactions with the glucose transporter Glut1 in human red cells adsorbed on lectin-gel beads.

The affinities of the human red cell glucose transporter Glut1 for D-glucose and cytochalasin B (CB) and the stoichiometry of CB binding vary with the Glut1 environment. In order to study the native state of Glut1 we adsorbed human red cells to wheat germ lectin agarose gel beads for frontal affinity chromatographic analyses. Glut1 showed relatively high affinities for D-glucose (Kd 12+/-1 mM) and CB (Kd 59+/-17 nM). The number of CB-binding sites per Glut1 monomer, 0.46+/-0.16, was approximately doubled upon coating the cells with polylysine, which induced cell association.

Biomembrane-affinity centrifugal analyses of solute interactions with membrane proteins.

We have developed a rapid centrifugal method for analyzing solute interactions with membrane proteins in cytoskeleton-depleted membrane vesicles or proteoliposomes sterically immobilized in Superdex 200 gel beads. The size and density of the gel beads allow fast sedimentation in a bench-top centrifuge. Biospecific interactions of cytochalasin B and D-glucose with the human red cell glucose transporter, Glut1, were analyzed. The binding constants and the molar ratio of inhibitor sites per protein monomer agreed well with recent results obtained by frontal affinity chromatography.

Immobilized biomembrane chromatography of highly lipophilic drugs.

Drug interaction with lipid bilayers was quantified by immobilized biomembrane chromatography on a series of columns containing different small amounts of human red cell membrane vesicles to extend and characterize this technique, which shows a potential for drug screening and prediction of drug absorption in humans. The chromatographic retention volume for each drug was essentially proportional to the amount of immobilized lipid, and the slope equalled the capacity factor (Ks) previously determined on single columns. Gel beds containing 0.5-2 micromol of membrane phospholipid allowed analysis of drugs with log Ks values of 2.5-4.3 in time periods of 1 min to 1 h. Highly lipophilic drugs could thus be analyzed conveniently in aqueous buffer.

Chromatographic approaches to liposomes, proteoliposomes and biomembrane vesicles.

Size-exclusion chromatography has been used for fractionation of liposomes, proteoliposomes and biomembrane vesicles of up to approximately 500 nm in size and for separation of these entities from smaller components. Liposome sizes, encapsulation stability, and solute affinities for membrane proteins have been determined. Counter-current distribution in aqueous two-phase systems has widened the range of applications to larger structures. Immobilized biomembrane vesicles and (proteo)liposomes provide stationary phases for chromatographic analysis of specific or nonspecific membrane-solute interactions.

Avidin-biotin immobilization of unilamellar liposomes in gel beads for chromatographic analysis of drug-membrane partitioning.

To construct a homogeneous lipid membrane chromatographic phase, biotinylated unilamellar liposomes of small and large sizes (SUVs and LUVs, respectively) were immobilized in avidin- or streptavidin-derived gel beads in amounts up to 55 micromol phospholipid/ml gel bed at yields above 50%. The immobilized liposomes exhibited excellent stability due to avidin-biotin multiple-site binding. The trapped volume and size distribution of the immobilized liposomes (0.33-0.42 microl/micromol lipid and 20-30 nm diameter for SUVs, 1.7-1.9 microl/micromol lipid and 80-120 nm for LUVs) indicated the unilamellarity and integrity of the immobilized liposomes. Partitioning of 15 pharmaceutical drugs into the bilayers of LUVs immobilized in different gel matrices correlated very well, as shown by chromatographic drug retention analysis. The partitioning of several beta-blockers into the immobilized LUVs showed a close correlation with their partitioning, reported in the literature, into free liposomes. The avidin-biotin-immobilized unilamellar liposomes can thus be used for chromatographic analysis and screening of solute-membrane interactions.

Biomembrane affinity chromatographic analysis of inhibitor binding to the human red cell nucleoside transporter in immobilized cells, vesicles and proteoliposomes.

The affinity of the human red cell nucleoside transporter for the transport inhibitor nitrobenzylthioinosine decreases upon protein purification. The affinity was highest for the whole cells (Kd, 0.04 nM), lowered upon cytoskeleton depletion (Kd, 0.2 nM) and lowest after partial purification and reconstitution (Kd, 0.3 nM), as determined by frontal affinity chromatography.

Evaluation of dynamic polar molecular surface area as predictor of drug absorption: comparison with other computational and experimental predictors.

The relationship between various molecular descriptors and transport of drugs across the intestinal epithelium was evaluated. The monolayer permeability (Pc) of human intestinal Caco-2 cells to a series of nine beta-receptor-blocking agents was investigated in vitro. The dynamic polar molecular surface area (PSAd) of the compounds was calculated from all low-energy conformations identified in molecular mechanics calculations in vacuum and in simulated chloroform and water environments. For most of the investigated drugs, the effects of the different environments on PSAd were small. The exception was H 216/44, which is a large flexible compound containing several functional groups capable of hydrogen bonding (PSAd,chloroform = 70.8 A2 and PSAd,water = 116.6 A2). The relationship between Pc and PSAd was stronger than those between Pc and the calculated octanol/water distribution coefficients (log Dcalc) or the experimentally determined immobilized liposome chromatography (ILC) retention. Pc values for two new practolol analogues and H 216/44 were predicted from the structure-permeability relationships of a subset of the nine compounds and compared with experimental values. The Pc values of the two practolol analogues were predicted well from both PSAd calculations and ILC retention studies. The Pc value of H 216/44 was reasonably well-predicted only from the PSAd of conformations preferred in vacuum and in water. The other descriptors overestimated the Pc of H 216/44 100-500-fold.

Freeze-thaw immobilization of liposomes in chromatographic gel beads: evaluation by confocal microscopy and effects of freezing rate.

Biological membranes immobilized in chromatographic gel beads constitute a multifunctional affinity matrix. Membrane protein-solute interactions and drug partitioning into the lipid bilayers can conveniently be studied. By the use of confocal laser-scanning microscopy (CLSM) the distribution of immobilized model membranes in the beads has been visualized for the first time. Freeze-thaw-immobilized liposomes in Superdex 200 gel beads were situated in a thick shell surrounding a liposome-free core. The amount of phospholipids immobilized by freeze-thawing was dependent on the temperature in the cooling bath and the type of test tube used. A bath temperature of -25 degrees C gave higher immobilization yield than freezing at -75 or -8 degrees C did. Freeze-thawing in the presence of liposomes did not affect the gel bead shape or the refractive index homogeneity of the agarose network of the beads, as shown by confocal microscopy.

Biomembrane affinity chromatographic analysis of nitrobenzylthioinosine binding to the reconstituted human red cell nucleoside transporter.

Solute interactions with membrane proteins can be analyzed by biomembrane affinity chromatography (BAC), previously applied to the human red cell glucose transporter. As a novel example, frontal BAC analysis of interactions between the nucleoside transport inhibitor nitrobenzylthioinosine (NBTI) and immobilized reconstituted nucleoside and glucose transporters from human red cells revealed two binding sites, presumably corresponding to the two transporters. The affinities and amounts of sites were determined by use of a double rectangular hyperbolic equation. The Kd value for NBTI binding to the nucleoside transporter in egg phospholipid proteoliposomes was 0.38 +/- 0.08 nM (22 degrees C, I = 0.16, pH 7.4), lower than previously reported for reconstituted systems. The molar ratio between the amounts of nucleoside transporter sites for NBTI and glucose transporter sites for cytochalasin B was 4.5 +/- 0.6%.

Chromatography on cells and biomolecular assemblies.

Red cells, biomembrane vesicles, proteoliposomes and liposomes non-covalently immobilized in gel particles or beads have been used as stationary phases for biomembrane affinity analyses and ion-exchange chromatographic separation. Lipid monolayers coupled to silica beads have been utilized for membrane protein purification in detergent solution and plant cell walls for group separation of macromolecules according to size and charge. Further methodological studies are essential to implement general practical application.