Per Lundahl

Immobilized-liposome chromatographic analysis of drug partitioning into lipid bilayers☆

The chromatographic retardation of drugs on a gel bed with immobilized liposomes was shown to correlate with the absorption of the drugs through epithelial cell layers, which is related to drug partitioning into the lipid bilayers of cell membranes. The capacity factors were divided by the lipid concentration (mM) in the gel bed to obtain specific capacity factors, Ks. The logarithm of the octanol-water distribution ratios showed a linear correlation with log Ks, whereas the logarithm of the apparent permeability coefficients in epithelial cell monolayers and the absorption of drugs orally administered in humans (data from other laboratories) increased over the interval 0 < log Ks < 1 and attained a saturation level in the interval 1 < log Ks < 3. Immobilized-liposome chromatography may be applicable for prediction of drug uptake through epithelial cell membranes.

Immobilized liposome chromatography of drugs for model analysis of drug-membrane interactions

Abstract Liposomes are composed of lipid bilayers surrounding aqueous compartments. For attempts to predict drug absorption through cell membranes by use of a chromatographic model system, liposomes or biological membrane vesicles can be sterically immobilized by entrapment in the pores of gel beads upon freeze-thaw fusion of small liposomes or vesicles. Alternatively hydrophobic ligands attached to the gel matrix can be used for immobilization. The chromatographic retention of a drug on a gel bed containing a known amount of liposomes or membrane vesicles reveals the degree of interaction between the drug and the lipid bilayers, after correction for drug-gel matrix interaction. The experiments are performed in aqueous buffer and the stability of liposome immobilization allows series of runs over periods of several weeks. Liposomal lipid composition and surface charge affect the interaction. The specific capacity factors of several drugs correlate reasonably well with drug permeability through Caco-2 epithelial cell monolayers and with absorption of orally administered doses in humans.

Immobilized liposome and biomembrane partitioning chromatography of drugs for prediction of drug transport

Drug partitioning into lipid bilayers was studied by chromatography on liposomes and biomembranes immobilized in gel beads by freeze–thawing. The drug retention volume was expressed as a capacity f ...

A model for ionic and hydrophobic interactions and hydrogen-bonding in sodium dodecyl sulfate-protein complexes

Abstract We propose a new hypothetical model for the structure of complexes between sodium dodecyl sulfate and proteins as described below: the detergent forms a flexible capped cylindrical micelle around which the hydrophilic segments of the polypeptide are helically wound. These polypeptide segments are attached by hydrogen bonds from the nitrogens of the peptide bonds to the sulfate oxygens of the detergent molecules. Cationic amino acid side-chains can also form ionic bonds with the sulfate groups. When the latter occurs the polypeptide assumes an α-helical conformation at the surface of the detergent cylinder in accordance with interpretations of circular dichroism measurements (Mattice, W.L. Riser, J.M. and Clark, D.S. (1976) Biochemistry 15, 4264–4272). An essentially regular arrangement of one hydrogen bond per peptide bond and two per dodecyl sulfate monomer is consistent with the known binding ratio of approximately one detergent molecule per two amino acid residues and with the proportionality between the polypeptide molecular mass or the number of residues and the length of the complex (Reynolds, J.A. and Tanford, C. (1970) J. Biol. Chem. 245, 5161–5165).The hydrogen-bonded structure of our model also agrees with the finding that dodecyl sulfate associates much more readily with proteins than does tetradecyltrimethylammonium chloride (Nozaki, Y., Reynolds, J.A. and Tanford, C. (1974) J. Biol. Chem. 249, 4452–4459). The axial length of the structure we propose can be estimated at approximately 0.6 A per amino acid residue. Hydrophobic polypeptide segments, including membrane-spanning α-helices of integral membrane proteins, can be accommodated in the interior of the elongated dodecyl sulfate micelle. Therefore, not only water-soluble proteins but also some integral membrane proteins may form the proposed type of complex. In the case of glycosylated membrane proteins the sugar moieties will protrude from the surface of the rod-shaped complex. We have named our model ‘the flexible helix’.

Crossed immunoelectrophoresis of human erythrocyte membrane proteins: Immunoprecipitation patterns for fresh and stored samples of membranes extensively solubilized with non-ionic detergents

Abstract 1. 1.|Human erythrocyte membranes (ghosts) were treated with four non-ionic detergents at pH 9.2 (5 °C) in a dilute buffer. More than 85% of the protein was solubilized. A protein concentration of up to 4 mg/ml was obtained. 2. 2.|The solubilized proteins were examined with rabbit antibodies against membrane material by crossed immunoelectrophoresis in agarose gels containing the solubilizing detergent. 3. 3.|The immunoelectrophoretic analyses showed that the membrane proteins were stable at −196 °C in the intact membrane. After solubilization with a non-ionic detergent changes occurred within two days at 5 °C but not at −20 °C. 4. 4.|A total of 19 membrane-specific immunoprecipitates were observed by crossed immunoelectrophoresis in the non-ionic detergent Berol EMU-043. Similar precipitation patterns were obtained with other non-ionic detergents. Some of the immunoprecipitates formed two peaks and some showed reaction of partial identity. 5. 5.|The antibodies precipitated most of the solubilized protein as demonstrated by immunoabsorption in an agarose gel of electrophoretically migrating antigens. 6. 6.|The precipitation pattern in crossed immunoelectrophoresis with a given antibody solution was reproducible with different membrane preparations. 7. 7.|Crossed immunoelectrophoresis in non-ionic detergents can be used for analysis of membrane protein fractions as a complement to polyacrylamide gel electrophoresis in dodecylsulfate.

Liposome chromatography: liposomes immobilized in gel beads as a stationary phase for aqueous column chromatography.

Liposomes have been used as a stationary phase for column chromatography with an aqueous mobile phase. They were immobilized in the pores of carrier gel beads by two methods: (A) hydrophobic ligands were coupled to the matrix of gel beads, which then were packed into a column and liposomes were applied and became associated with the ligands by hydrophobic interaction; and (B) phospholipids and detergent were dialysed in the presence of gel beads; many of the liposomes that formed in the pores of the beads were sterically immobilized by the gel matrix. Proteoliposomes containing red cell glucose transport protein in the lipid bilayers were immobilized in a column by method A. This column retained D-glucose longer than L-glucose. In contrast to L-glucose, D-glucose was transported into and out of the immobilized liposomes, causing an increased retention. Liposomes with (stearylamine)+ or (phosphatidylserine)- in their lipid bilayers were immobilized by method B and the gel beads were packed into a column. A protein of opposite charge was applied in excess. Under suitable conditions, the protein molecules became close-packed on the liposome surfaces. Ion-exchange chromatographic experiments with proteins showed that these sterically immobilized liposomes were also stable enough to be used as a stationary phase. The loss of lipids was 5-23% in the first run at high protein load and with sodium chloride gradient elution but was lower in subsequent runs. It is proposed that water-soluble molecules can be separated and their interactions with liposome surfaces studied by chromatography on immobilized liposomes in detergent-free aqueous solution. Membrane proteins can be inserted and ligands can be anchored in the lipid bilayers for chromatographic purposes.

Isoelectric focusing in free Ampholine solution and attempts at isoelectric focusing in pH gradients created in ordinary buffers.

In isoelectric focusing of proteins according to the method of Svenssonlv 2 and Vesterberg and Svenssons a stable pH gradient is created by electrophoretic migration of carrier ampholytes toward stationary positions. The solution is usually stabilized against convection by a density gradient of sucrose, or by a gel of polyacrylamide or agarose.4 Any anticonvectional agent can, however, interact with the proteins to be studied and thus cause a series of undesirable phenomena; in addition, the isoelectric points as well as the measured pH values may be influenced by the presence of sucrose or any other stabilizing medium. Such considerations motivated us to develop further the previously described technique of free isoelectric focusing in a slowly revolving electrophoresis tubes (the rotation of the tube counteracts convective disturbances). \ Not only the supporting medium, but also the carrier ampholytes can interact with proteins.6 Indications of artifacts due to such interaction have been reported.’ Furthermore, Ampholine gives rise to disturbing uv background (see Reference 17 and FIGURE 6a-c) and tends to precipitate during the focusing in free solution (FIGURE 15). Therefore, we have tried to focus proteins in ordinary buffer solutions (in the absence of Ampholine), creating a pH gradient either by means of a temperature gradient,

Improved preparation of the integral membrane proteins of human red cells, with special reference to the glucose transporter

or by means of electrophoresis in a concentration gradient of a neutral substance, such as sucrose, sucrose polymerized with epichlorohydrin (Ficoll). or polyacrylamide gels.

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

Human red cell membranes were isolated and partially stripped of peripheral proteins by gel filtration of hemolysates on a Sepharose CL-4B column at pH 8 connected in tandem to a Sepharose CL-6B column at pH 10.5. The eluted material was washed by centrifugations, once at pH 10.5 and twice at pH 12. In this way, water-soluble proteins and peripheral membrane proteins were thoroughly removed, and 0.2 g of integral membrane proteins could be prepared within 10 h from 0.2 litre of red cells. The exposure to high pH did not lower the D-glucose transport activity, and electrophoretically pure glucose transport protein could be isolated from this preparation. Gel filtration in sodium dodecyl sulfate separated the integral membrane components into four fractions, one of them containing 4.5-material; gel electrophoresis showed about 14 zones and two-dimensional electrophoresis resolved up to 100 mostly minor components, among which the glucose transporter focused around pH 7. However, purified glucose transporter focused around pH 8. Glucose and nucleoside transport proteins were co-purified in active form on DEAE-cellulose and a fraction isolated by adsorption to Mono Q was used for immunization of mice and production of monoclonal antibodies. One hybridoma produced antibodies that reacted with material in the 4.5-region, possibly the glucose transport protein, and not with band 3-material. Upon two-dimensional electrophoresis of integral membrane components that had been solubilized with octyl glucoside the immunoreactive and the silver-stained 4.5-material focused in a broad range from pH 6 to pH 9. A possible explanation for this heterogeneity might be interaction between the glucose and nucleoside transport proteins and negatively charged lipids.

19. Detergent-Containing Gels for Immunological Studies of Solubilized Erythrocyte Membrane Components

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.