Phospholipase A(2)-catalyzed membrane leakage studied by immobilized liposome chromatography with online fluorescent detection

Unilamellar liposomes composed of phosphatidylcholine with an entrapped self-quenching fluorescent dye, calcein, were immobilized in chromatographic gel beads by avidin-biotin binding. Bee venom phospholipase A(2) (PLA(2)) was applied in a small amount onto the immobilized liposome column. The release of calcein from the immobilized liposomes resulting from the catalyzed hydrolysis of the phospholipids was detected online by immobilized liposome chromatography (ILC) using a flow fluorescent detector. The PLA(2)-catalyzed membrane leakage of the immobilized liposomes as studied with ILC was found to be affected by the gel pore size used for immobilization, by liposome size, and as expected by the concentration of calcium, but was unaffected by the flow rate of ILC. The largest PLA(2)-induced calcein release from the liposome column was detected on large unilamellar liposomes immobilized on TSK G6000PW or Sephacryl S-1000 gel in the presence of 1 mM Ca(2+) in the aqueous mobile phase. Comparison with the PLA(2)-catalyzed membrane leakage in free liposome suspensions, we conclude that the fluorescent leakage from liposomes hydrolyzed by PLA(2) can be rapidly and sensitively detected by ILC runs using large amount of immobilized liposomes with entrapped fluorescent dye.     

Partitioning of triphenylalkylphosphonium homologues in gel bead-immobilized liposomes: chromatographic measurement of their membrane partition coefficients

Unilamellar liposomes of small or large size, SUVs and LUVs, respectively, were stably immobilized in the highly hydrophilic Sepharose 4B or Sephacryl S-1000 gel beads as a membrane stationary phase for immobilized liposome chromatography (ILC). Lipophilic cations of triphenylmethylphosphonium and tetraphenylphosphonium (TPP+) have been used as probes of the membrane potential of cells. Interaction of TPP+ and triphenylalkylphosphonium homologues with the immobilized liposomal membranes was shown by their elution profiles on both zonal and frontal ILC. Retardation of the lipophilic cations on the liposome gel bed was increased as the hydrophobicity of the cations increased, indicating the partitioning of lipophilic cations into the hydrocarbon region of the membranes. The cations did not retard on the Sepharose or Sephacryl gel bed without liposomes, confirming that the cations only interact with the immobilized liposomes. Effects of the solute concentration, flow rate, and gel-matrix substance on the ILC were studied. The stationary phase volume of the liposomal membranes was calculated from the volume of a phospholipid molecule and the amount of the immobilized phospholipid, which allowed us to determine the membrane partition coefficient (KLM) for the lipophilic cations distributed between the aqueous mobile and membrane stationary phases. The values of KLM were generally increased with the hydrophobicity of the solutes increased, and were higher for the SUVs than for the LUVs. The ILC method described here can be applied to measure membrane partition coefficients for other lipophilic solutes (e.g., drugs).     

Evaluation of temperature and guanidine hydrochloride-induced protein–liposome interactions by using immobilized liposome chromatography

Hydrophobic interactions between nine model proteins and net-neutral lipid bilayer membranes (liposomes) under stress conditions were quantitatively examined by using immobilized liposome chromatography (ILC). Small or large unilamellar liposomes were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and immobilized in a gel matrix by utilizing covalent coupling between amino-containing lipids and activated gel beads or avidin–biotin biospecific binding. Retardation of bovine carbonic anhydrase (CAB) in ILC was pronounced at particular temperatures (50 and 60 °C) where the local hydrophobicity of theses protein molecules becomes sufficiently large. Protein-induced leakage of a hydrophilic dye (calcein) from immobilized liposomes interior was also drastically enhanced at particular temperatures where large retardation was observed. For other proteins examined, similar results were also observed. The specific capacity factor of the proteins characteristic for the ILC and the amount of calcein released from immobilized liposomes were successfully expressed as a function of the product of the local hydrophobicities of proteins and liposomes, regardless of protein species and the type of the stress conditions applied (denaturant and heating). These findings indicate that lipid membranes have an ability to non-specifically recognize local hydrophobicities of proteins to form stress-mediated supramolecular assemblies with proteins, which may have potential applications in bioprocesses such as protein refolding and separation. ILC was thus found to be a very useful method for the quantitative detection of dynamic protein–liposome interactions triggered by stress conditions.     

Avidin–biotin-immobilized liposome column for chromatographic fluorescence on-line analysis of solute–membrane interactions

Unilamellar liposomes with entrapped fluorescent dye calcein were stably immobilized in gel beads by avidin–biotin-binding. The immobilized liposomes remained extremely stable upon storage and chromatographic runs. The immobilized calcein-entrapped liposomes were utilized for fluorescent analysis of solute–membrane interactions, which in some cases are too weak to be detected by chromatographic retardation. A liposome column was used as a sensitive probe to detect the interactions of membranes with pharmaceutical drugs, peptides and proteins. Retardation of the solutes was monitored using a UV detector. Perturbation of the membranes, reflected as leakage of the entrapped calcein by some of the solutes, can thus be detected on-line using a flow-fluorescent detector. For the amphiphilic drugs or synthetic peptides, perturbation of membranes became more pronounced when the retardation (hydrophobicity) of the molecules increased. On the other hand, in the case of positively-charged peptides, polylysine, or partially denatured bovine carbonic anhydrase, significant dye leakage from the liposomes was observed although the retardation was hardly to be measured. Weak protein–membrane interactions can thus be assumed from the large leakage of calcein from the liposomes. This provides additional useful information for solute–membrane interactions, as perturbation of the membranes was also indicated by avidin–biotin-immobilized liposome chromatography (ILC).     

Development of metal affinity-immobilized liposome chromatography and its basic characteristics

Metal affinity-immobilized liposome chromatography (MA-ILC) was newly developed as a chromatographic technique to separate and analyze peptides. The MA-ILC matrix gel was first prepared by immobilizing liposomes modified with functional ligands. The functional ligand used to adsorb metal ions was N-hexadecyl iminodiacetic acid (HIDA), which is obtained by attaching a long alkyl chain to an iminodiacetic acid (IDA). Cu(II) ion was first adsorbed on the gel matrix through its complex formation with the HIDA on the surface of the immobilized liposome. Synthetic peptides of various types ranging in size from 5 to 40 residues were then used, and their retention properties on the MA-ILC were evaluated. The retention property of peptides on the MA-ILC by using a usual imidazole elution was compared with the retention property in the case of the immobilized metal affinity chromatography (IMAC) and an immobilized liposome chromatography (ILC). It was found that the retention property of peptides on the MA-ILC has the features of both the IMAC and the ILC; the retention ability of peptides depends on both the number of histidine residues in peptides and the liposome membrane affinity of the peptides. Histidine and tryptophan residues among amino acid residues in peptides indicated a high contribution coefficient for the peptide retention on the MA-ILC, probably due to their metal ion and membrane interaction properties, respectively.     

Conformationally changed cytochrome c-mediated fusion of enzyme- and substrate-containing liposomes.

The fusion between enzyme-containing liposomes and substrate-containing liposomes was studied, utilizing conformationally altered cytochrome c as fusion mediator under stress conditions. The liposomes were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and liposome aggregation and subsequent liposome fusion were induced by the addition of cytochrome c, which was partially denatured by 0.5 M guanidinium hydrochloride (GuHCl). In the presence of 0.5 M GuHCl, cytochrome c was found to have a significantly large local hydrophobicity which was determined with the aqueous two-phase partitioning method. Under these conditions, cytochrome c could efficiently bind to POPC bilayer membranes as quantitatively evaluated by immobilized liposome chromatography (ILC). The retardation of cytochrome c treated with 0, 0.5, and 1 M GuHCl on ILC could be correlated with the corresponding local hydrophobicity of cytochrome c. The enzymatic reaction triggered by liposome fusion involved the proteolytic enzyme alpha-chymotrypsin and its substrate succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-AAPF-pNA), which were separately trapped in POPC liposomes. Addition of partially denatured cytochrome c (most likely in the molten globule state) to the mixture of enzyme- and substrate-containing liposomes resulted in the release of one of the hydrolysis products, p-nitroaniline, to the outer phase of the fused liposomes, indicating that the enzymatic reaction occurred during the liposome fusion process. Such a coupled fusion-reaction system may have specific advantages over the conventional fusion analysis and may find application as drug delivery system.     

Optimal preparation of immobilized liposome-bound cellulase for hydrolysis of insoluble cellulose in an external loop airlift bioreactor

Immobilized liposome-bound cellulase (ILC) was optimally prepared for the ILC-catalyzed hydrolysis of insoluble cellulose in an external loop airlift bioreactor. The liposomes with mean diameters of 200, 100, and 50 nm were used to prepare three kinds of ILCs, i.e., ILC(200), ILC(100) and ILC(50), respectively. The activity and stability of ILC(100) were examined with soluble cellulose (CMC) in addition to the insoluble substrate of cellulose powder (CC31) in a shaking flask as well as the airlift bioreactors. The experiments were carried out with 45 degrees C and pH 4.8 being found to be optimal for the activity. The activity of ILC(100) was stable in either airlift or shaking flask bioreactor during the five times repeated hydrolyses of CC31 corresponding to a total reaction time of 240 h. This confirmed that the cellulase molecules were covalently bonded to the liposomes covalently bound to the chitosan gel beads. Nevertheless, the activity of ILC(100) with CMC steadily decreased throughout the repeated reactions, suggesting an adverse effect of CMC on the ILC(100) activity. Among the three ILCs, ILC(50) was found to be the most stable and productive biocatalyst during the repeated hydrolyses of insoluble CC31 in the airlift bioreactor. More than 70% of the initial activity of ILC(50) was retained even after the six times repeated reactions for 288 h. Conversely, the ILC(200) was found to be the most unstable catalyst. Such a difference in stability among these ILCs was suggested to be caused by the difference in physical stability of their liposome membranes to the liquid shear stress due to the rising bubbles and circulating liquid as well as that in the amount of the cellulase molecules unstably incorporated in the membranes. ILC(50) was thus shown to have the most potential for an efficient hydrolysis of insoluble cellulose in a practical airlift bioreactor.     

Steroid bioconversion in water-insoluble organic solvents: Δ1-Dehydrogenation by free microbial cells and by cells entrapped in hydrophilic or lipophilic gels

A cell suspension in a water-insoluble organic solvent (benzene: n-heptane, 1 : 1 by volume) of Nocardia rhodocrous (previously induced to synthesize steroid Δ¹dehydrogenase) rapidly catalyzed the stoichiometric oxidation of 4-androstene-3,17-dione (4-AD) to androst-l,4-diene-3,17-dione (ADD) in the presence of phenazine methosulfate (PMS). High levels of 4-AD or PMS reduced the conversion rates. No appreciable decrease in the conversion rate was observed on adding aqueous buffer solution to the thawed ceils (up to 9.4 g water/g dry cell). The whole cells were immobilized by entrapment in a hydrophilic gel (H-gel) or a lipophilic gel (L-gel) by use of a water-soluble or water-insoluble photocrosslinkable prepolymer. The reticula of H- and L-gel matrices were impregnated with water and organic solvent, respectively. Both the H- and L-gels could convert 4-AD to ADD in the presence of PMS, the L-gel showing a slightly higher conversion rate. Various lines of evidence indicate that the limiting factor is the penetration rate of 4-AD into gel particles for the H-gel, and the penetration rate of PMS for the L-gel. The catalytic activities decreased considerably after several successive runs with the free cell suspension system, while the immobilized cells were more stable, the stability of H-gel and L-gel being almost the same.     

Characterization and immobilization of liposome-bound cellulase for hydrolysis of insoluble cellulose

The liposome-bound cellulase was prepared by covalently coupling cellulase with the enzyme-free liposomes bearing aldehyde groups so that cellulase was located solely on the outer membrane of liposomes. The modified cellulase possessed the higher activity efficiency and lipid-based specific activity than the cellulase-containing liposomes reported previously. The enzyme-free liposomes bearing aldehyde groups were covalently immobilized with the chitosan gel beads and the free cellulase was coupled with the treated gel beads to prepare the immobilized liposome-bound cellulase. The activity efficiency of the immobilized liposome-bound cellulase was much higher than that of the conventionally immobilized cellulase. The results on reusability of the immobilized liposome-bound cellulase in the hydrolysis of either soluble or insoluble cellulose showed that the immobilized liposome-bound cellulase had the higher remaining cellulase activity and reusability than the conventionally immobilized cellulase for the hydrolysis of either type of cellulose. The liposomal membrane was suggested to be efficient in maintaining the cellulase activity during the hydrolysis.     

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.     

New type of glucose sensor based on enzymatic conversion of gel volume into liquid column length

A way to convert the volume change of a biochemo-mechanical gel into the change in liquid column length was developed. Our trial sensor device consisted of a small compartment for incorporating the gel, a flow channel with a filled dye solution, and a poly(dimethylsiloxane) (PDMS) diaphragm by which the gel and the dye solution were separated. A lightly cross-linked N-isopropylacrylamide (NIPAAm)/acrylic acid (AA) copolymer gel with immobilized glucose oxidase was used as a sensing element. It was found that a change in the gel volume caused by the immobilized enzyme reaction was accurately converted into a change of the column length (Deltal) with the help of the PDMS diaphragm. By use of a cylindrical gel (diameter approximately 2 and thickness approximately 1 mm), the time curve of Deltal varied depending upon glucose concentration over a range of 0.2-50 mM; in particular, it is of importance that semilogarithmic plots of Deltal (in mm) against glucose concentration (in mM) can be used as a calibration curve. For glucose solutions of mM order, 1 min was enough to determine the concentrations, whereas 10 min was required for concentrations of microM order. When the measurement time was limited within 10 min, the lower detection limit was 200 microM. The response was affected by buffering capacity of the samples, but this was controllable through reduction of the sample volume. These results indicate that the present way can be used for the determination of glucose concentration.     

Continuous removal of Cr(VI) from aqueous solution catalysed by palladised biomass of Desulfovibrio vulgaris

Growth-decoupled cells of Desulfovibrio vulgaris NCIMB 8303 can be used to reduce Pd(II) to cell-bound Pd(0) (Bio-Pd(0)), a bioinorganic catalyst capable of reducing hexavalent chromium to less toxic Cr(III), using formate as the electron donor. Magnetic resonance imaging showed that Bio-Pd(0), immobilized in chitosan and agar beads, is distinguishable from the surrounding gel and is evenly dispersed within the immobilization matrix. Agar-immobilized Bio-Pd(0) and 'chemical Pd(0)' were packed into continuous-flow reactors, and challenged with a solution containing 100 microM Cr(VI) (pH 7) at a flow rate of 2.4 ml h(-1). Agar-immobilized chemical Pd(0) columns lost Cr(VI) reducing ability by 160 h, whereas columns containing immobilized Bio-Pd(0) maintained 90% reduction until 680 h, after which reduction efficiency was gradually lost.     

Covalent immobilization of unilamellar liposomes in gel beads for chromatography

For immobilized (proteo)liposome chromatography, unilamellar liposomes were covalently bound within gel beads that had been activated by CNBr, N-hydroxysuccinimide, tresyl, or chloroformate. Liposomes composed of phosphatidylcholine (PC) and 2 mol% of amino-containing lipid (phosphatidylethanolamine-caproylamine) were immobilized in the activated gels at 5-35 micromol lipid/ml gel and yields of 11-70%. The highest immobilized amount was found in chloroformate-activated TSK G6000PW gel, which contains large pore size (>100 nm). Liposomes composed of PC alone could also be attached to the chloroformate-activated gels at 33-42 micromol/ml gel and yields of 58-65%, probably by crosslinking of the phosphate moiety of phospholipid with the active group of the adsorbent. Liposomes prepared by various phospholipids with or without amino-containing lipids can generally be immobilized in the chloroformate-activated gels. The covalently bound liposomes were characterized by their high stability, unilamellarity, permeability of the membranes, and drug-membrane partition properties. A stable membrane phase was constructed for chromatographic experiments to be performed under extreme elution conditions.     

Catalytic activity of poly(acryloyl morpholine)-immobilized horseradish peroxidase in organic/aqueous solvent mixtures

The immobilization of horseradish peroxidase by covalent coupling within an expanded poly(acryloyl morpholine) gel network is described. The activity of the immobilized horseradish peroxidase was compared with that of the native enzyme in aqueous buffer and in buffered mixtures of dimethyl-formamide/water, ethanediol/water, methanol/water and tetrahydrofuran/water of varying solvent ratios at pH 6.1. On increasing the organic solvent concentration in the substrate solution, active immobilized enzyme retained its activity much better than an equivalent amount of the native enzyme. The oxidation of ferrocene (water-insoluble) and ferrocene derivatives to the corresponding ferricinium ions, was accomplished efficiently by the immobilized enzyme in buffered 50% methanol/water solution. The immobilized enzyme exhibited superior resistance to thermal denaturation.     

Continuous production of bacitracin by immobilized living whole cells of Bacillus sp.

Whole cells of Bacillus sp., a bacitracin-producing bacteria, were immobilized in polyacrylamide gel. The continuous production of bacitracin by an immobilized whole-cell-containing air-bubbled reactor was examined with 0.5% peptone solution. The bacitracin productivity (28 units/ml/hr) obtained with this system was higher than that with a batch system. The effluent bacitracin concentration increased with increasing aeration rate and reached a steady-state maximum above the aeration rate of 3.0 liter/min. A high bacitracin productivity was retained for at least eight days when the gel was washed with sterilized saline at a flow rate of 250 ml/hr for 2 hr once a day. The half-life of the immobilized whole-cell system was about 10 days. Bacitracin productivity by the immobilized whole-cell reactor was higher than that by a conventional continuous fermentation process at high dilution rates.     

A simple model for DNA elution from filters

DNA chain scission, induced both in vitro and in vivo by various agents, is an event of great biological relevance. The damage is currently evaluated by empirical membrane separation techniques; the results are quite reproducible and the sensitivity higher than 1 single strand break per 10(9) Daltons. We outline a simple theory of the filtration of coiled macrosolutes, having a random size distribution, through porous membranes, considered as being in quasi-steady flow. The basic transport equation Jj = cj (1 - sigma)Jv is solved by considering that the value of sigma j, the reflection coefficient of component j, (1 less than or equal to j less than or equal to N), is given by (1 - KjRj), where Kj is the partition constant between pore and solution, a function of the conformational entropy loss of the coil, and Rj accounts for the frictional force experienced by a particle moving along the pore. The problem of evaluating the volume Vs filled up with solute has been approached according to a simplified theory of the excluded volume for flexible polymers; the result is Vs = sigma nj4/3 pi(rGj)3 where rGj is the jth radius of gyration. The solution of the resulting set of N differential equations gives nj, the number of molecules of component j remaining on the filter, as a function of the elution volume V. The theory demonstrates that the process is governed by the average dimensions of the coil, so affording a universal calibration of filter elution methods, in excellent agreement with the experiments.     

Basic Studies for Continuous Production of L-Aspartic Acid by Immobilized Escherichia coli Cells

By using a column packed with immobilized Escherichia coli cells entrapped in a polyacrylamide gel lattice, conditions for continuous production of L-aspartic acid from ammonium fumarate were investigated. When a solution of 1 M ammonium fumarate (pH 8.5) containing 1 mM Mg(2+) was passed through the immobilized cell column at a flow rate of space velocity (SV) = 0.8 at 37 C, the highest rate of reaction was attained. From the column effluents, L-aspartic acid was obtained in good yield. The immobilized cell column was very stable.     

On-column refolding of denatured lysozyme by the conjoint chromatography composed of SEC and immobilized recombinant DsbA

DsbA (disulfide bond formation protein A) located in the periplasm of Escherichia coli is a disulfide isomerase, which is vital to disulfide bonds formation directly affecting the nascent peptides folding to the correct conformation. In this paper, recombinant DsbA was firstly immobilized onto NHS-activated Sepharose Fast Flow gel. Then Sephadex G-100 gel was sequentially packed on the top of recDsbA Sepharose Fast Flow, and a so-called conjoint chromatography column composed of SEC and immobilized recombinant DsbA was constructed. Denatured lysozyme was applied on the conjoint column. The effect of SEC volume, flow rate, loading amount and volume, pre-equilibrium mode and KCl concentration in the buffer on lysozyme refolding were investigated in detail and the stability of DsbA immobilization was evaluated. Finally the reusability of the conjoint refolding column was also tested. When loading 2.4 mg denatured lysozyme in 0.5 ml solution, the activity recovery reached 92.7% at optimized experimental conditions, and the conjoint column renaturation capacity decreased only 7.7% after six run reuse due to the use of SEC section in the chromatographic refolding process. The conjoint chromatography offers an efficient strategy to refold proteins in vitro with high productivity and column reusability.     

Enzyme, β-galactosidase immobilized on membrane surface for galacto-oligosaccharides formation from lactose: Kinetic study with feed flow under recirculation loop

The work focuses on producing galacto-oligosaccharides (GOS) through an enzymatic reaction with lactose under a partial recirculation loop by utilizing membrane-immobilized β-galactosidase. Cross-linking through covalent bonding, using gluteraldehyde, was employed to immobilize enzyme on a microporous polyvinylidene fluoride membrane. GOS synthesis was carried out in a laboratory fabricated reaction cell, whereby three immobilized membranes were housed in series. The reaction was conducted at varying initial lactose concentrations (ILCs) and feed flow rates at pH 6 and 40 °C. A maximum GOS of 30% (dry basis) was obtained after 60 h of reaction time, 50 g/L ILC, 241 U of enzyme (specific loading of 600 U/g-membrane), and 0.5 mL/min of feed flow rate at 56% lactose conversion. The GOS yield increased with increased ILC and decreased feed flow rate. The selectivity of GOS formation increased by increasing both the ILC and the feed flow rate, whereas the reverse was true for mono-saccharides. The immobilized enzyme retained ∼50% of its initial activity after 30 days of storage at 20 °C, while the native enzyme lost 100% of its activity within 21 days. Furthermore, a five-step, nine-parameter model was developed, and simulated results showed excellent agreement with the experimental data.     

A novel flow cytometric assay to quantify interactions between proteins and membrane lipids

A diverse set of experimental systems has been developed to probe protein-lipid interactions. These include measurements with the headgroups of membrane lipids in solution, immobilized membrane lipids, and analysis of protein binding to membrane lipids reconstituted in liposomes. Each of these methodologies has strengths but also substantial limitations. For example, measurements between proteins and lipid headgroups or with immobilized membrane lipids do not probe interactions in their natural environment, the lipid bilayer. The use of liposomes, however, was so far mostly restricted to biochemical flotation experiments that do not provide quantitative and/or kinetic data. Here, we present a fast and sensitive flow cytometric method to detect protein-lipid interactions. This technique allows for quantitative measurements of interactions between multiple fluorescently labeled proteins and membrane lipids reconstituted in lipid bilayers. The assay can be used to quantify binding efficiencies and to determine kinetic constants. The method is further characterized by a short sampling time of only a few seconds that allows for high-content screening procedures. Finally, using light scatter measurements, the described method also allows for monitoring changes of membrane curvature as well as tethering of liposomes evoked by binding of proteins.