Ultrafiltration Cell Research Articles

Protein charge ladders: a new technique for studying electrostatic interactions in ultrafiltration systems

Although, previous studies have demonstrated the importance of electrostatic interactions in membrane systems, there are few techniques available for the accurate analysis of these effects. This manuscript describes a novel approach to study electrostatic interactions during ultrafiltration using protein charge ladders, which consist of a series of chemical derivatives of a given protein that differ by single charge units. Charge ladders were formed by reacting myoglobin and bovine carbonic anhydrase with acetic anhydride to block free lysine groups. The concentration and net electrical charge of the individual “rungs” were determined by capillary electrophoresis. Sieving experiments were performed using a simple stirred ultrafiltration cell. The results clearly show an increase in electrostatic exclusion of the more negatively-charged species in low ionic strength solutions. The membrane surface charge density was estimated from myoglobin sieving data by comparing the results to model calculations. This surface charge density was in good agreement with independent measurements of the membrane zeta-potential. These results demonstrate that protein charge ladders can be used to study electrostatic interactions in much the same way as polydisperse dextrans have been used to study steric interactions in membrane systems.

CDNA Cloning, Heterologous Expression and Characterization of Anti-Neuroexcitation Peptides (ANEPS) of Scorpion Buthus martensii Karsch.

INTRODUCTION. The neurotoxins from scorpion venoms are specifically active on mammals or insects, and divided into two main categories based on their molecular sizes and pharmacological activities. The short-chain toxins consist of 30 to 40 amino acid residues with 3 or 4 disulfide bonds and predominantly act upon potassium or chloride channels, meanwhile the toxins contain 60 to 70 amino acid residues, the long-chain toxins, with 4 disulfide bonds primarily influence sodium channels. These toxins can selectively bind to the corresponding channels on the membrane of excitable cells, impair the initial rapid depolarization phase of the action potential in nerve and muscle, thus result in neurotoxicity. In China, scorpions and their tissues or extracts have been extensively used for treatment of apoplexy, epilepsy, convulsion, facial paralysis, hemiplegia and anti-inflammation etc in traditional Chinese medicine for over thousands of years. Neurotoxins of the scorpion Buthus martensii Karsch (Bmk), a representative species in China and east Asia, have been widely studied in recent fifteen years, including studies on their primary and three-dimensional structures, their cDNA and genomic genes, their physiological and biological functions, and their pharmaceutical properties. We cloned a new anti-neuroexcitation peptide cDNA, expressed and purified it in E. coli and characterized its biological activities. The anti-neuroexcitation peptide is highly homologous to the previously reported anti-epilepsy peptide and shows strong calcium channel enhancement activity. METHOD. According to the previously reported N-terminal amino acid sequence of antiepilepsy peptide (1), two degenerate primers were designed and synthesized. Total RNA was isolated from scorpion telsons and reverse transcribed. RACEs were performed. The PCR product was cloned and sequenced. The putative mature anti-neuroexcitation peptide (ANEP) encoding cDNA was then cloned into pET28a expression plasmid. Recombinant ANEP was purified by His-Bind affinity column. The resulting elute was desalted and concentrated with Amicon stirred ultrafiltration cells (1kDa membrane). Then the protein was further purified on Superdex 30 chromatography and analyzed by SDS-PAGE. Anti-neuroexcitation bioassay was performed essentially according to Xu et al (2) by using thiosemicarbazide induced mice convulsion model. Physiologic saline was used as blank control, sodium phenobarbital (4.0mg/kg) as positive control and recombinant anti-neuroexcitation peptide (2.0mg/kg) as sample respectively. The delayed time on the seizure of convulsive symptom in ANEP group and positive group, in comparison with the control group, was expressed in percentage of the control group and was used as indicator of anti-neuroexcitation biological activity. The cDNA sequence has been submitted to GenBank under accession number AF122003.

Ultrafiltration of a waste emulsified cutting oil using organic membranes

The influence of operating conditions on permeate flux was investigated using organic ultrafiltration membranes to treat a synthetic cutting-oil emulsion containing 0.05 to 0.8 wt. % oil. A commercial cutting oil, Alba-Kool, and four organic ultrafiltration membranes, two made of polysulfone (PM10 and PM30) and the other two of regenerated cellulose (YM10 andYM30), with cut-off sizes of 10 000 or 30 000 Dalton, were used.All experiments were performed in a commercially available stirred ultrafiltration cell. Within the range of operating conditions studied, it was found that the highest fluxes wereobtained at the following conditions: crossflow velocity (rotation speed) of 375 rpm, temperature of 40 °C andtransmembrane pressure of 0.1–0.15 MPa. Oil rejections between99 and 99.9% were obtained. Complete membrane regeneration wasachieved with a 2 vol. % aqueous solution of a commercial cleaning agent (Derquim+, Panreac).

An archaeal chaperonin-based reactor for renaturation of denatured proteins.

We describe an original chaperonin-based reactor that yields folded and active proteins from denatured materials. We used the 920-kDa chaperonin of the archaeon Sulfolobus solfataricus, which does not require any protein partner for its full activity and assists in vitro folding with low substrate specificity. The reactor consists of an ultrafiltration cell equipped with a membrane that retains the chaperonin in a functional state for folding in solution and permits the flowthrough of the folded substrates. By studying the ATP-dependent functional cycle of the chaperonin, we were able to use the reactor for repeated refolding processes. The scale-up of the reactor is made possible by the overproduction of chaperonin in Sulfolobus solfataricus cells that acquired thermotolerance upon appropriate heat shock.

Hindered convection of proteins in agarose gels

The hindered convection of macromolecules in gels was examined by measuring the sieving coefficient (Θ, the ratio of filtrate to retentate concentration) of globular proteins in agarose membranes, as a function of protein size and gel concentration. The proteins used were lactalbumin (Stokes–Einstein radius, r s = 2.1 nm), ovalbumin ( r s = 3.0 nm), and BSA ( r s = 3.6 nm), and the volume fraction of agarose ( φ) was varied from 0.04 to 0.08. Agarose membranes were prepared on polyester mesh supports and studied in a stirred ultrafiltration cell. The Darcy permeabilities of the gels were determined in addition to Θ, and separate measurements of the mass transfer coefficient in the stirred cell were made to correct Θ for the effect of concentration polarization. The values of Θ decreased with increasing r s or φ, as expected. From the measurements of Θ and estimates of the protein diffusivity and equilibrium partition coefficient, the convective hindrance factor ( K c) was calculated for each protein–gel combination. This is the ratio of the average solute velocity (in the absence of diffusion) to the superficial fluid velocity. For small r s and φ it was found that K c slightly exceeded unity, whereas K c < 1 at larger values of r s or φ. This behavior was qualitatively, but not quantitatively, consistent with predictions from existing hindered transport theories for media consisting of parallel fibers or straight pores. Given evidence from previous partitioning and diffusion data that an agarose gel is better represented as a randomly oriented array of fibers, the large quantitative discrepancies between the data and models are not entirely surprising. Thus, the present results suggest that there is a need to extend theories of hindered convection to random arrays of fibers.

Ultrasound effect on cross-flow filtration of polyacrylonitrile ultrafiltration membranes

Ultrasound effects on permeate flux and rejection of solute were studied by using two types of polyacrylonitrile (PAN) ultrafiltration (UF) membranes reinforced with non-woven cloth. The membrane was set in cross-flow UF cell and the filtration was carried out by permeating 1 wt% dextran solutions under 30 kPa until steady-state flux was obtained. Then, ultrasound with 45 kHz of frequency was irradiated on the UF cell in water. It was found that the ultrasound irradiation significantly increased the permeate flux of different molecular weight (MW) dextran solutions. Evidence was presented that ultrasound irradiation was effective to enhance the membrane permeate flux for dextran solution.

Ultrafiltration of Divalent Metal Cations from Aqueous Solution Using Polycarboxylic Acid Type Biosurfactant

Biosurfactant-based ultrafiltration was used to remove the divalent metal ions, Cu+2, Zn+2, Cd+2, and Ni+2ions from aqueous solution containing either a single metal species or a mixtures of metal ions. Polycarboxylic acid type biosurfactant, sodium salt of 2-(2-carboxyethyl)-3-decyl maleic anhydride (DCMA-3Na) derived from spiculisporic acid was added to trap the metal ions through the binding onto the micelles and/or the formation of metal-biosurfactant precipitates. The present study aimed at investigating the permeating flux and the removal efficiencies of divalent metal cations from aqueous solutions using a batch-type stirred ultrafiltration cell. Concerning the flux decline, the membrane of molecular weight cutoff (MWCO) 1,000 is more vulnerable to the formation of a gel layer and the concentration polarization than that of MWCO 3,000. Formation of metal-biosurfactant precipitates makes the gel concentration of DCMA-3Na relatively low compared with the case of chemically synthesized surfactant such as SDS and CPC. However, biosurfactant-based ultrafiltration is of practical significance because high rejection efficiencies for cupric, zinc, cadmium ions can be achieved only with equimolar ratio of DCMA-3Na to the metal ions. DCMA-3Na exhibits the metal binding affinity on the order of Cd+2> Cu+2≈ Zn+2> Ni+2when one metal species is present. Even under the competitive binding condition when four metal ions are present in the mixture, DCMA-3Na shows the same affinity order toward the metal ions, although small variations in rejection coefficients are observed.

Optimization of plasma proteins concentration by ultrafiltration

This paper describes the optimization of the operation of an ultrafiltration cell filtering plasma proteins on the basis of the economic criteria of Shen and Probstein. The optimum hydrodynamic parameters were determined on a pilot ultrafiltration unit which was equipped with either an organic tubular PCI BX6 with 20 000 daltons molecular weight cut-off or mineral Ceraver P 19–40 membranes with an average pore radius size of 500 Å. Optimum parameters corresponding to average transmembrane pressures of 3 and 4 bar and tangential flow rates of 5.4 and 0.5 m/s for Ceraver and PCI membranes, respectively, were established. The organic PCI BX6 membrane appeared more efficient than the ceramic Ceraver membrane. Experimental data for fouling and rejection coefficient are presented. High apparent rejection coefficients of the Ceraver membrane attained indicated that a dynamic membrane was formed which altered the molecular weight cut-off of the virgin membrane. The potential uses of the process for pollution control applications and cleaning of the ultrafiltration membranes are presented.

Modeling of ultrafiltration process for a two-component aqueous solution of low and high (gel-forming) molecular weight solutes

Filtration of sucrose from a mixture of sucrose and poly(vinyl alcohol) (PVA) was studied in a stirred ultrafiltration cell. PVA formed a gel layer and was completely rejected by the membrane. Two membranes of different molecular weight cut-off, namely, 1 K and 10 K were chosen. By the former, sucrose was partially retained and by the latter, it was completely permeable in the absence of PVA. A mathematical model was formulated for simultaneous prediction of permeate flux and observed rejection of sucrose. The characteristics of the gel layer and other relevant parameters of the model were determined by independent sets of experiments. It was observed that the steady-state flux was independent of pressure and equal to the flux of the gel-forming component alone (in the absence of permeating solute). Profiles of permeate flux and observed rejection of sucrose with time agreed well with the experimental observations.

Measurement and modeling of nitric oxide release rates for nitric oxide donors.

An accurate model of the nitric oxide (NO)-release rate is essential for predicting the temporal NO-release rate and resulting NO concentrations for NO donors. Knowledge of the NO-release rate and/or the NO concentration is beneficial for assessing the physiological or pathological effects of NO on cell systems. This study describes a method to measure the temporal NO-release rate from NO donor compounds utilizing a modified ultrafiltration cell. For this study, the NO-release rates of spermine NONOate and diethylamine NONOate were measured and kinetically modeled at pH 7.4 and 37 degrees C. An advantage of this method is that complete dissolution of the NONOate was not necessary for modeling the NO-release rate. One model parameter, which is the number of moles of NO released per mole of decomposed NONOate, is 1.7 +/- 0.1 and 1.5 +/- 0.2 for spermine and diethylamine NONOate, respectively. The other model parameter, which is the NONOate first-order decomposition rate constant, is 0.019 +/- 0.002 min-1 and 0.47 +/- 0.10 min-1 for spermine and diethylamine NONOate, respectively, as determined from NO concentration profiles. The decomposition rate constant measured by spectrophotometry was consistent with the above value for spermine NONOate but was approximately half the above value for diethylamine NONOate. Preliminary experiments using spectrophotometry showed that for both NONOates the decomposition activation energy was approximately 100 kJ/mol. The NO-release rate model for spermine NONOate was applied to a model for predicting the NO concentration in oxygenated solution. The NO concentration was measured in phosphate buffer, culture medium, and Tyrode's solution. Excellent agreement was observed between experimental and predicted NO concentrations.

Hindered transport of macromolecules in isolated glomeruli. II. Convection and pressure effects in basement membrane

The filtration rates for water and a polydisperse mixture of Ficoll across films of isolated glomerular basement membrane (GBM) were measured to characterize convective transport across this part of the glomerular capillary wall. Glomeruli were isolated from rat kidneys and the cells were removed by detergent lysis, leaving a preparation containing almost pure GBM that could be consolidated into a layer at the base of a small ultrafiltration cell. A Ficoll mixture with Stokes-Einstein radii ranging from about 2.0 to 7.0 nm was labeled with fluorescein, providing a set of rigid, spherical test macromolecules with little molecular charge. Filtration experiments were performed at two physiologically relevant hydraulic pressure differences (delta P), 35 and 60 mmHg. The sieving coefficient (filtrate-to-retentate concentration ratio) for a given size of Ficoll tended to be larger at 35 than at 60 mmHg, the changes being greater for the smaller molecules. The Darcy permeability also varied inversely with pressure, averaging 1.48 +/- 0.10 nm2 at 35 mmHg and 0.82 +/- 0.07 nm2 at 60 mmHg. Both effects could be explained most simply by postulating that the intrinsic permeability properties of the GBM change in response to compression. The sieving data were consistent with linear declines in the hindrance factors for convection and diffusion with increasing pressure, and correlations were derived to relate those hindrance factors to molecular size and delta P. Comparisons with previous Ficoll sieving data for rats in vivo suggest that the GBM is less size-restrictive than the cell layers, but that its contribution to the overall size selectivity of the barrier is not negligible. Theoretical predictions of the Darcy permeability based on a model in which the GBM is a random fibrous network consisting of two populations of fibers were in excellent agreement with the present data and with ultrastructural observations in the literature.

Significance of duration and amplitude in transmembrane pressure pulsed ultrafiltration of binary protein mixtures

This study examines the effects of varying the pulse amplitude and duration on permeate flux and solute throughput for a binary protein solution in crossflow transmembrane pressure pulsed ultrafiltration with semi-permeable hydrophobic membranes. Solutions of 1% BSA and 0.3% IgG in 0.15 M NaCl, with pH 7.4 were used in a crossflow ultrafiltration cell with a crossflow shear rate of 700 s −1, pulsing frequency of 0.5 Hz, and an average operating pressure of 66 kPa was used. Polysulfone membranes with 100 000 MWCO were used throughout the study. Average pulse amplitude was varied between 60 and 27 kPa and the average pulse duration ranged between 0.13 and 0.34 s of backpressure per second of operation. The results showed that variations in pulse duration did not significantly effect permeate flux. However, mass flux of BSA was enhanced due to pulsing by as much as a factor of three. Only the case which combined reduced pulsing pressure and short duration resulted in negligible deviation in BSA concentration from the nonpulsed case results. One proposed mechanism for this is that transmembrane pressure pulsing may improve solute flux by removing lodged solute in the pores. The backward flow potentially carries the previously lodged solute out away from the pore, clearing the pore for solute transport in the next ultrafiltration cycle.

Flux prediction of black liquor in cross flow ultrafiltration using low and high rejecting membranes

Experimental studies were carried out to observe the flux decline behaviour and rejection characteristics of kraft black liquor in a rectangular cross flow ultrafiltration cell. A theoretical model was developed to predict flux and real rejection for osmotic pressure controlled ultrafiltration. Predicted values were compared with the experimental results. Standard macromolecules (PEG-6000 and dextran-18 600) were also used for comparison. Growth of the concentration boundary layer, variation of the concentration profile across the length of the channel and estimation of the membrane surface concentration at different cross flow velocities were studied through the developed model. The membrane used for the standard solutes gave rejection values for black liquor in the range of 18 to 40%. This range was improved by using high rejecting membrane (85–92%). Successive filtration through both the membranes were also carried out (using the permeate of low rejecting membrane as feed to the high rejecting) and gave a rejection range of 91–95%. These experimental values were also compared with the model predictive results. The results obtained from the low rejecting membrane showed excellent agreement; however, discrepancies were observed and explained for the results from high rejecting membranes, either direct or successively used.

DNA Structure Determines Protein Binding and Transcriptional Efficiency of the Proenkephalin cAMP-responsive Enhancer

Two precisely arranged proenkephalin cAMP response elements (CREs) behave as a single protein binding site. The experiments described support a model in which a secondary structural change creates a new binding site, which is made up of sequences from both of the CREs. The CRE-binding protein (CREB) binds CRE-1, but binding there is entirely dependent on the presence of CRE-2. Electron spectroscopic images show that a CREB dimer occupies twice as much DNA in the proenkephalin gene as in the prodynorphin gene. The enhancer region is sensitive to P1 nuclease in a CREB concentration-dependent manner, and sensitivity is strand-specific, indicating protein-stabilized structural change. DNase I analysis shows that in the native proenkephalin gene, CREB binds both CRE-1 and CRE-2. In vivo, both CREs are occupied in the transcriptionally active proenkephalin gene, while neither is in the silent gene. Whereas CREB can bind CRE-2, mutation or elimination of either proenkephalin CRE alters response to second messengers and transcription factors. Thus, binding to CRE-2 alone is not sufficient. Specific and efficient transcription of the proenkephalin gene requires the presence of both CREs, precisely arranged to allow them to form a single protein binding site.

Ultrafiltration recovery of polyvinyl alcohol from desizing wastewater

Abstract Recovery of polyvinyl alcohol (PVA) by ultrafiltration (UF) from simulated desizing wastewater is investigated. Batch experiments using an UF cell were conducted to investigate the effects of operating pressure, temperature and mixer speed on the PVA retention, filtration rate and the rejection coefficient. It was observed in the experimental results that an operating pressure over 2 bars is sufficient for efficient operation of the PVA recovery process. Operating temperature lower than 80°C was seen to significantly reduce the efficiency of PVA recovery. Similar effect was also found for the mixer operating at a speed below 500 rpm. In general, the UF process was observed to be quite effective, capable of over 96% retention efficiency of PVA from the dilute desizing wastewater.

The Concentration Polarization Effect of a Macroligand on Affinity Ultrafiltration

The concentration polarization effect of a macroligand on the affinity ultrafiltration of an important glycopeptide antibiotic, vancomycin, was studied. The affinity ultrafiltration was carried out in a stirred cell, and the system was operated in a circulating and once-through mode. The macroligand was prepared by coupling the ligand D-alanyl-D-alanine to a water soluble polymer, dextran. The concentration polarization of dextran itself did not show any effect on vancomycin retention in the ultrafiltration cell. On the other hand, the retention of vancomycin was enhanced in the presence of concentration polarization of the macroligand on the membrane surface. The enhancement was due to the affinity binding of vancomycin to the macroligand in the concentration polarization layer. The observed affinity binding behaviors of vancomycin in the affinity ultrafiltration systems were in good agreement with that predicted by the thin film model coupled with the Langmuir isotherm and fast binding kinetics.

Production of human immunodeficiency virus by chronically infected cells grown in protein-free medium.

A human T cell line chronically infected with Human Immunodeficiency Virus (HIV) has been adapted to grow in a chemically defined, protein-free medium. Virus particles are produced at rates comparable to those of serum-supplemented cultures; virus preparations free of undesirable proteins can be produced in preparative amounts by simple ultrafiltration procedures and cell culture supernatants can be used as such for the preparation of ELISA solid phases. This material has been used very conveniently for studies concerning characterization of antibodies against HlV-specific proteins, interaction of HIV with complement components and inclusion of human cell-derived proteins into virions; we propose its use as a powerful tool for the structural as well as functional analysis of the virus particle itself.

Operational patterns affecting lactic acid production in ultrafiltration cell recycle bioreactor

Lactic acid production with cell recycling on an ultrafiltration tubular membrane reactor was studied; higher lactic acid concentrations as well as productivities were obtained under long-term fermentations compared with other high cell density systems. Different operational conditions, namely dilution rates and start-up modes, were assessed. Performances were very different at the three different dilution rates tested (D = 0.20 h(-1), D = 0.40 h(-1), or D = 0.58 h(-1)). The different behaviours are discussed and factors responsible for them are presented. The best way to operate for lactic acid production is chosen, the dilution rate of D = 0.40 h(-1) being the one providing the best overall performance. On the other hand, results show that of the two start-up modes tested, continuous start (membrane open) permits higher permeabilities throughout the operational runs than batch start (membrane closed). Operational stability was found to be directly associated with membranes that work at "steady state," the membrane permeability being kept around 15 L/m(2) h. Optimized cell bleed can improve time of operation if such membrane permeability can be maintained for a longer time. A comparison of results with those obtained in other lactic acid production systems is presented; such comparison shows that this tubular ultrafiltration membrane cell recycle reactor presents three important advantages: (1) concomitant lactic acid concentrations and productivities; (2) long periods of operation at reasonable permeabilities; and (3) good mechanical stability permitting the use of steam sterilization. (c) 1995 John Wiley & Sons, Inc.

Production of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine by entrapped ACV-synthetase from Streptomyces clavuligerus.

delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine (ACV)-synthetase from Streptomyces clavuligerus was studied under conditions that enabled the reuse of the enzyme. Coupling of ACV-synthetase to DEAE-Trisacryl and aminopropyl-glass resulted in an immobilized enzyme product of little or no catalytic activity. However, an enzyme reactor was designed by physical confinement of partially-purified ACV-synthetase in an ultrafiltration cell. This system was stimulated by phosphoenolpyruvate at lower concentrations of ATP, an effect not observed with purified enzyme. Up to 30% conversion of the limiting substrate, cysteine, to ACV occurred under semi-continuous conditions. Reaction products were investigated as potential inhibitors: AMP was the most inhibitory, but only when used at concentrations in excess of those produced in reaction mixtures. Under a nitrogen atmosphere, both product and enzyme stabilities were greatly improved and the enzyme retained 45-65% of its initial activity after five uses at room temperature during a 24-h period. Extrapolations based on these data suggest that 1.3 g partially purified enzyme (0.13 U g-1) would be capable of producing 411 mg of ACV in a 1-L reaction mixture in this period.

Development of concentration profile and prediction of flux for ultrafiltration in a radial cross-flow cell

The limiting flux phenomena in ultrafiltration were studied. The experiments were carried out using an asymmetric membrane in a radial cross-flow cell. The permeate flux was predicted for black liquor, a paper plant effluent, in comparison to the results using a standard macromolecular solution of poly(ethylene glycol) (PEG 6000). The governing equations for the simulation of radial cross-flow ultrafiltration cell were solved. The differential equations were coupled with an osmotic pressure model and they were solved successively. The fluxes were predicted employing two different techniques. The first technique was an approximate solution using a simplified velocity field and a similarity transformation, called here the simplified approach. The second technique was a detailed simulation using perturbation theory and orthogonal collocation, the so-called detailed approach. The overall average fluxes obtained by the two approaches were compared. The results showed agreement between the two approaches and with the experimental data.