Fast protein chromatofocusing of human very-low-density lipoproteins

Using fast protein chromatofocusing, a high-efficiency column chromatography method with a self-generated pH gradient and focusing effects, soluble human very-low-density lipoprotein (VLDL) apolipoproteins were fractionated between pH 6.3 and 4.0. In the presence of 6 mol/l urea and with a flow rate of 1 ml/min, one run (up to 10 mg of protein) took 30 min. VLDL apolipoproteins were separated in seven peaks. As revealed by SDS-polyacrylamide gel electrophoresis, isoelectric focusing and double-immunodiffusion against mono-specific antisera, fractions corresponded to the following proteins: apolipoprotein C-I, albumin, apolipoproteins A-I, E, C-II plus C-III0, C-III1 and C-III2, respectively. Apolipoproteins were eluted in sharp, well-resolved peaks. The recovery of proteins was 78% of the starting material. With fast protein chromatofocusing, an efficient isolation of single apolipoproteins is possible from small amounts of VLDL apolipoprotein preparations. This technique is superior to the commonly used, time-consuming methods for apolipoprotein isolation.     

Chromatofocusing of peptides and proteins using linear pH gradients formed on strong ion-exchange adsorbents

Although it is commonly believed that a column packing used for chromatofocusing must have an "even" buffering capacity in order to produce a linear pH gradient, it is demonstrated here that linear pH gradients suitable for chromatofocusing can be produced on a column packing having a minimal buffering capacity. In particular, if either a strong-acid cation-exchange column packing or a strong-base anion-exchange column packing is presaturated with either a weak acid titrated with a strong base, or a weak base titrated with a strong acid, respectively, to the initial pH, then a linear or nearly linear pH gradient can be formed using a polyampholyte elution buffer by taking advantage of the presence of small quantities of weak-acid or weak-base functional groups that generally exist on these types of column packings. Experimental and theoretical studies are used to demonstrate that such systems have potential advantages over traditional chromatofocusing methods in terms of the speed of the separation, the resolution achieved, and the range of applications possible. Among other techniques described, a method for separating tryptic peptides using chromatofocusing and a strong-acid cation-exchange column packing is demonstrated to be a useful alternative to capillary isoelectric focusing and ion-exchange chromatography using a salt gradient for this purpose.     

Ammonia/potassium exchange in methanogenic bacteria

Methanospirillum hungatei exposed to ammonia in a K+-free buffer lost up to 98% of the cytoplasmic K+ through an ammonia/K+ exchange reaction. The exchange was immediate, and occurred in cells poisoned by air or by other metabolic inhibitors. Additions of NH4OH or various NH+4 salts (or methylamine) were most effective in causing K+ depletion in media of alkaline pH, suggesting that NH3 was the chemical species crossing the membrane. In alkaline media, the exchange reaction resulted in a dissipation of the transmembrane pH gradient (inside acidic), but had only small effects on the membrane potential until concentrations of ammonia were used above those required to abolish the K+ gradient. Through the use of NH4Cl to vary the cytoplasmic pH at a constant acidic external pH, and NH4OH to abolish the transmembrane pH gradient at various alkaline external pH values, we conclude that methanogenesis is sensitive to both the pH of the cytoplasm and the medium. Methanogenesis in Msp. hungatei and Methanosarcina barkeri was inhibited dramatically at external pH values more acidic than 6.5 or more alkaline than 7.5. Dramatic K+ depletion in response to ammonia additions at pH 8.0 occurred with Ms. barkeri, another strain of Msp. hungatei, Escherichia coli, and Bacillus polymyxa. In several other methanogens, ammonia/potassium exchange was hardly detected.     

Separation of manganese peroxidase isoenzymes on strong anion-exchange monolithic column using pH-salt gradient

Different chromatographic methods including chromatofocusing are used for separation of manganese peroxidase (MnP) isoforms and their isolation from the fungal growth medium. We tested strong anion exchange methacrylate based monolithic columns as a stationary phase for fast separation of MnP's. Sodium acetate buffers of two different pH values (6 and 4) were used for formation of reproducible pH gradient. The entire cycle, involving analysis and column regeneration, was completed in 3 min. Use of pH gradient showed better MnP isoform separation comparing to the salt gradient, while application of combined pH-salt gradient, resulted in further improvement.     

Plasmonic Optical Trapping in Biologically Relevant Media

We present plasmonic optical trapping of micron-sized particles in biologically relevant buffer media with varying ionic strength. The media consist of 3 cell-growth solutions and 2 buffers and are specifically chosen due to their widespread use and applicability to breast-cancer and angiogenesis studies. High-precision rheological measurements on the buffer media reveal that, in all cases excluding the 8.0 pH Stain medium, the fluids exhibit Newtonian behavior, thereby enabling straightforward measurements of optical trap stiffness from power-spectral particle displacement data. Using stiffness as a trapping performance metric, we find that for all media under consideration the plasmonic nanotweezers generate optical forces 3–4x a conventional optical trap. Further, plasmonic trap stiffness values are comparable to those of an identical water-only system, indicating that the performance of a plasmonic nanotweezer is not degraded by the biological media. These results pave the way for future biological applications utilizing plasmonic optical traps.     

Purification of recombinant green fluorescent protein using chromatofocusing with a pH gradient composed of multiple stepwise fronts

Green fluorescent protein (GFP), which fluoresces in the green region of the visible spectrum and is widely used as a reporter for gene expression and regulation, was overexpressed in the JM105 strain of Escherichia coli transformed with pBAD-GFP. A two-step chromatofocusing procedure was used to purify GFP starting from cell lysate, with each step employing a pH gradient extending from pH 5.5 to 4.0. The first chromatofocusing step was performed using a low-pressure column in which a retained stepwise pH front formed by adsorbed buffering species was used to capture GFP directly from clarified cell lysate and selectively focus it into a chromatographic band. The second step utilized a high-performance column under mass overloaded conditions where a similar pH front acted as a protein displacer and led to the formation of a highly concentrated rectangular band of GFP. The overall procedure yielded a 50-fold increase in purity, a 20-fold volume reduction, and a recovery and purity for GFP of 60% and 80%, respectively. Because the method employs a strong-base ion-exchange column packing and low-cost buffers formed with formic and acetic acids instead of the proprietary column packings and polyampholyte elution buffers more generally used for chromatofocusing, it appears to be a practical alternative for the preparative ion-exchange chromatography of GFP in particular and for the recovery of recombinant proteins from cell lysate in general. A discussion is also given concerning the choice of appropriate buffers for the rational design of pH gradients involving retained, stepwise pH fronts that span a given pH range and of the use of the fluorescence properties of GFP for flow visualization and chromatographic process development.     

Preparative purification of B-phycoerythrin from the microalga Porphyridium cruentum by expanded-bed adsorption chromatography

B-Phycoerythrin (B-PE) is a major light-harvesting pigment of microalgae. Due to its high fluorescence efficiency and its intense and unique pink color, it is widely used as a fluorescent probe and analytical reagent as well as being employed as a natural dye in foods and cosmetics. Tedious methodologies for B-PE purification have been published. In this work we present a new, fast, preparative and scaleable two-step chromatographic method for B-PE purification from the red microalga Porphyridium cruentum. Initially, phycobiliproteins were released from the microalga cells by osmotic shock and captured by applying the centrifuged cell suspension to a column containing 74 ml Streamline-DEAE equilibrated with 50 mM acetic acid-sodium acetate buffer, pH 5.5, using expanded-bed adsorption chromatography at an upward flow of 200 cm h(-1). After adsorption, washing was carried out in the expanded-bed mode. Having removed unbound proteins and cellular debris, the bed was allowed to sediment and a B-PE-rich solution was eluted with a downward flow of the same 250 mM buffer. In order to obtain pure B-PE, we utilized conventional ion-exchange chromatography with a column of DEAE-cellulose loaded directly with the eluate from Streamline-DEAE and developed using a discontinuous gradient of acetic acid-sodium acetate buffer, pH 5.5. With this new methodology, 66% of B-PE contained in the biomass of the microalgae was recovered, a value significantly higher than those obtained following other methodologies. The B-PE purity was tested using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and spectroscopic characterization.     

A Rapid Purification of L-Glutamic Acid Decarboxylase from the Brain of the Locust Schistocerca gregaria

L-Glutamic acid decarboxylase (GAD; EC 4.1.1.15) was purified to apparent homogeneity from the brain of the locust Schistocerca gregaria using a combination of chromatofocusing (Mono P) and gel filtration (Superose 12) media. The homogeneity of the enzyme preparation was established by native polyacrylamide gel electrophoresis (PAGE) with silver staining. The molecular weight of the purified enzyme was estimated from native gradient gel electrophoresis and gel filtration chromatography to be 97,000 +/- 4,000 and 93,000 +/- 5,000, respectively. When analysed by sodium dodecyl sulphate-PAGE, the enzyme was found to be composed of two distinct subunits of Mr 51,000 +/- 1,000 and 44,000 +/- 1,500. Tryptic peptide maps of iodinated preparations of these two subunits showed considerable homology, suggesting that the native enzyme is a dimer of closely related subunits. The purified enzyme had a pH optimum of 7.0-7.4 in 100 mM potassium phosphate buffer and an apparent Km for glutamate of 5.0 mM. The enzyme was strongly inhibited by the carbonyl-trapping reagent aminooxyacetic acid with an I50 value of 0.2 microM.     

Capture of human monoclonal antibodies from a clarified cell culture supernatant by phenyl boronate chromatography

In this work, we investigated the feasibility of using phenyl boronate (PB) chromatography for the direct capture of monoclonal antibodies from a CHO cell supernatant. Preliminary results, using pure protein solutions have shown that PB media can bind to human antibodies, not only at strong alkaline conditions but also at acidic pH values. In fact, antibodies have been found to bind in the pH range 5.5-8.5. On the other hand, insulin and human serum albumin did not bind at alkaline pH but at lower pH, which reflects the importance of non-specific interactions with the matrix. Different binding and eluting buffers were evaluated for the capture of immunoglobulin G (IgG) from a CHO cell supernatant and the most promising results were obtained using 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid at pH 8.5 as binding buffer and 1.5 M Tris-HCl as eluting buffer. Using a step elution, all IgG was recovered in the elution pool with a maximum purification factor of 56. A gradient elution allowed a further increase of the final purity, yet achieving a slightly lower yield. IgG recovery was around 85% and the purification factor was 76. The highest purity was obtained when the pH of the cell supernatant feed was previously adjusted to 8.5. Starting from an initial protein purity of 1.1% and high-performance liquid chromatography (HPLC) purity of 2.2%, after PB adsorption, a final protein purity of 85% and a HPLC purity of 88% was achieved.     

Determination of the intravesicular pH of fragmented sarcoplasmic reticulum with 5,5-dimethyl-2,4-oxazolidinedione.

The intravesicular pH (pHi) of fragmented sarcoplasmic reticulum (SR) of the skeletal muscle was determined from the distribution of 5,5-dimethyl-2,4-oxazolidinedione (DMO), a weak organic acid, between the intra- and extravesicular spaces. The pHi's thus obtained were found to be slightly lower (0.02-0.17 pH unit) than the pH's of the external medium (pHe) at pH 6.5-8.5 in the presence of 105 mM KCl and 40 mM Tris-maleate buffer. The higher the pHe, the greater the pH gradient. When pHe was changed, pHi attained equilibrium within about 20 min, the time necessary for the separation of the SR by centrifugation. When 0.25 M sucrose and 5 mM Tris-maleate buffer were used instead of 105 mM KCl and 40 mM buffer, the pH gradient increased to 0.56. It was also demonstrated by direct measurements of pHe with a glass-electrode pH meter that K+ ions added to the external medium exchanged the intravesicular H+ ions. From these results it appears that the pH gradient across the SR membrane was at the Donnan equilibrium. In this state, the Donnan potentials corresponding to pH gradients of 0.17 and 0.56 were -9.3 and -30.6 mV, respectively.     

Determination of erythrocyte transit times through micropores. I--Basic operational principles

To study the transit times of each red blood cell passing through cylindrical micropores and in order to evaluate sub-population of cells with regard to their deformability, we have developed a new system called the cell transit time analyser (CTTA). By using an AC voltage (100 KHz) across a special filter, we measure the electrical conductance change produced by the cells passing through the pores under a known driving pressure. This computer based device provides the distribution of transit times tau for 2000 cells in 1 minute and as a result the mean transit time [tau]. Experiments with red cells were designed to evaluate the flow behavior of both normal cells and cells whose mechanical properties were artificially altered. Cell volume was changed by use of non-isotonic media. Cell shape and cell volume were modified by varying the pH of the suspending buffer. Results of these experiments are: 1) a skew distribution of transit times towards high tau values for both control cells and artificially altered cells is observed: 2) [tau] is minimum for isotonic conditions and increases sharply for either hypotonic or hypertonic media: 3) [tau] is minimum at physiological pH and increases for either acid or alcaline changes of pH.     

Studies on phosphoribosylpyrophosphate synthetase from Giardia intestinalis.

A substantial degree of purification, up to 3200-fold, with recoveries of 8-11% of phosphoribosylpyrophosphate (P-Rib-PP) synthetase from Giardia intestinalis extracts was achieved by the high resolution techniques of anion exchange chromatography and chromatofocusing columns on a fast protein liquid chromatography system. A Mono P chromatofocusing column gave rise to an enzyme peak eluting from the column at pH 4.5, indicating that the enzyme has an isoelectric point (pI) at approximately this value. The molecular weight of the enzyme was found to be 150,000 from a Sephacryl S-200 column. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified enzyme gave a single protein band with a subunit molecular weight of 38,000, indicating that the enzyme existed as a tetramer. The properties of G. intestinalis P-Rib-PP synthetase in terms of pH optimum, isoelectric point, subunit structure, phosphate requirement, metal and nucleotide specificity, appear to be very similar to those of the enzyme from other sources.     

Energy coupling to nitrate uptake into the denitrifying cells of Paracoccus denitrificans

This study deals with the effects of the agents that dissipate the individual components of the proton motive force (short-chain fatty acids, nigericin, and valinomycin) upon the methyl viologen-coupled nitrate reductase activity in intact cells. Substitution of butyrate or acetate for chloride in Tris-buffered assay media resulted in a marked inhibition at pH 7. In a Tris--chloride buffer of neutral pH, the reaction was almost fully inhibitable by nigericin. Alkalinisation increased the IC(50) value for nigericin and decreased the maximal inhibition attained. Both types of inhibitions could be reversed by the permeabilisation of cells or by the addition of nitrite, and that caused by nigericin disappeared at high extracellular concentrations of potassium. These data indicate that nitrate transport step relies heavily on the pH gradient at neutral pH. Since the affinity of cells for nitrate was strongly diminished by imposing an inside-positive potassium (or lithium) diffusion potential at alkaline external pH, a potential dependent step may be of significance in the transporter cycle under these conditions. Experiments with sodium-depleted media provided no hints for Na(+) as a possible H(+) substitute.     

A Simple Method for Density Gradient Zonal Electrophoresis with Imidazole-Glycine Buffer and its Application to a Study of Cyclic Amp-Binding Protein

A system of sucrose density gradient electrophoresis with an imidazole-glycine buffer is described which allows the use of a simple apparatus without separate buffer reservoirs, electrode chambers and bridges. Changes in pH during electrophoresis are held to a minimum. The procedure is easy and capable of high resolution. An example of the application of this method to a preparation of a cyclic AMP binding protein is shown.     

Role of bicarbonate/CO2 buffer in the initiation of vesicle-mediated calcification: mechanisms of aortic calcification related to atherosclerosis

Calcifying vesicles play an important role in the mechanism of aortic calcification induced by dietary cholesterol interventions. This study was initiated to test the hypothesis that alterations in the ratio of bicarbonate/CO2, which is a main physiological buffer, could affect vesicle-mediated calcification. Using rabbits as a model, in vitro calcification of vesicles isolated from aortas was performed to study the effect of the bicarbonate buffer on the mineralization process. When Tris buffer was initially used to maintain pH of the media, ATP-dependent vesicle calcification increased with pH of calcifying media. By replacing Tris with physiological bicarbonate/CO2 buffer, ATP-dependent vesicle calcification increased rapidly with increased ratios of bicarbonate/CO2. The increase appears to be a result of elevated levels of pH through the alteration in the ratios of bicarbonate/CO2. The effect of the physiological concentration of bicarbonate (30 mM) on pH of calcifying media was remarkable since 50 mM of Tris buffer at pH 7.6 failed to prevent a rapid rise in pH under atmospheric CO2. The effect of bicarbonate and CO2 on vesicle calcification was dependent on the ratio of the surface area to the volume of calcifying media, since the ratio profoundly affects the exchange rate between the gas and liquid phases of CO2. Although the pathological conditions that alter the pH remain unknown, it is conceivable that blockage in the supply of blood CO2 to the media by intimal thickening in the lesions could contribute to focal calcification. We conclude that bicarbonate buffer could provide a dynamic and rapid transitional increase in pH of extracellular fluids, thereby creating a favorable condition for the initiation of vesicle-mediated calcification under pathological conditions.     

pH gradients generated by polyprotic buffers. II. Experimental validation

The experimental validation refers to the computer program reported in the companion paper, able to simulate the course of pH, buffering power (beta) and ionic strength (I) of polyprotic buffers (either singly or in a mixture) titrated over any pH range. With simple oligoamines (up to five nitrogens) it is shown that it is impossible to generate linear pH gradients in the pH 4-10 interval, unless they are mixed in appropriate ratios. With pentaethylene hexamine, when used alone, it is possible to create a linear pH 4-10 interval, provided the molarity ratios are altered in the two chambers of the gradient mixer. The general rule operating for generation of linear pH intervals is constancy of buffering power throughout the titration. Local minima of beta produce steeper gradients, while local beta maxima flatten it. The ideal delta pK to arrange for linear pH gradients during titration is centred around 1 pH unit; thus polyprotic buffers with very large delta pK values (e.g., EDTA) appear to be totally useless for this purpose. The present computing algorithms should be quite efficient for optimizing existing buffer recipes for chromatofocusing or ampholyte displacement chromatography or for creating new, properly tailored, buffer mixtures.     

Effect of pH on the control release of microencapsulated dye in lecithin liposomes. II

The objective of our work has been the microencapsulation of dyes with lecithin from soybean, with the formation of liposomes, as a substitute for synthetic auxiliaries so as to improve the quality of the effluent. Current scenarios promote the disintegration and leakage of the liposomes, such as, changes in temperature, pH, and the use of surfactants. Since dyeing process is a mix of all these parameters, we pretended to study each one separately. Changes in pH at constant temperature induce a release of dye similar with changes in temperature. In acid conditions, we found a very fast initial dye release which doesn't occur in basic conditions. Using carboxyfluorescein, as a pH fluorescence probe, we concluded that the liposome membrane doesn't protect the liposome interior from changes on the external pH.     

A general method for the extraction of citrus leaf proteins and separation by 2D electrophoresis: a follow up

With the aim of studying differentially expressed proteins as a function of abiotic and biotic stress in citrus plants, we optimized a protocol for the extraction of total leaf proteins and their 2-DE separation using commercially available immobilized pH gradient strips (IPGs) in the first dimension. Critical factors for good reproducibility of citrus leaf protein separation were identified: trichloroacetic acid (TCA)/acetone precipitation after extraction in lysis buffer, sample fractionation on narrow range overlapping IPGs and sample-cup loading at the anodic or cathodic end of the strip. The use of thiourea and a strong detergent (C7BzO) in the solubilization/rehydration buffer, coupled with the increase to 10% of SDS in the equilibration buffer before the second dimension seemed to affect positively the resolution of basic proteins. Using our protocol we resolved about 30 basic proteins on 6.3-8.3 pH range strips. Further, our protocol was successfully applied reproducibly on the analysis of control and salt exposed leaf samples of Citrus reshni Hort. Ex Tan.     

Energy coupling to nitrate uptake into the denitrifying cells of Paracoccus denitrificans

This study deals with the effects of the agents that dissipate the individual components of the proton motive force (short-chain fatty acids, nigericin, and valinomycin) upon the methyl viologen-coupled nitrate reductase activity in intact cells. Substitution of butyrate or acetate for chloride in Tris-buffered assay media resulted in a marked inhibition at pH 7. In a Tris-chloride buffer of neutral pH, the reaction was almost fully inhibitable by nigericin. Alkalinisation increased the IC₅₀ value for nigericin and decreased the maximal inhibition attained. Both types of inhibitions could be reversed by the permeabilisation of cells or by the addition of nitrite, and that caused by nigericin disappeared at high extracellular concentrations of potassium. These data indicate that nitrate transport step relies heavily on the pH gradient at neutral pH. Since the affinity of cells for nitrate was strongly diminished by imposing an inside-positive potassium (or lithium) diffusion potential at alkaline external pH, a potential dependent step may be of significance in the transporter cycle under these conditions. Experiments with sodium-depleted media provided no hints for Na⁺ as a possible H⁺ substitute.     

The isolation and characterization of specific 3-methylcholanthrene-binding proteins from rat liver cytosol

The major proteins to which 3-methylcholanthrene specifically binds have been purified over 480-fold with a 45% yield compared to a rat liver 100,000g supernate. The procedure involved a batch ion-exchange technique together with hydrophobic gel filtration and chromatofocusing chromatography. The multiple, specific 3-methylcholanthrene-binding proteins obtained from this protocol had apparent isoelectric points of pH 6.3, 6.0, 5.7, and 5.5 on elution from a chromatofocusing column. They all shared a common sedimentation coefficient as determined by sucrose gradient analysis of 4.4 S. Gel filtration on Sephadex G-75 gave a common Stokes radius of 27 A. An analysis of these chromatofocusing peaks by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed those which eluted at pH 6.3 and 6.0 to contain two major protein bands of Mr 32,000 and 34,000, together with several contaminating proteins. In contrast, the peaks from chromatofocusing which eluted at pH 5.7 and 5.5 contained three major proteins of Mr 40,000, 25,000, and 14,000. The specific binding capability of these chromatofocusing peaks was found to be unstable to temperatures of -30 degrees C and below. Competition studies showed that these proteins were not steroid receptors, and that only polycyclic aromatic hydrocarbons which could induce cytochrome P-450c were able to displace 3-methylcholanthrene from the binding site. A marked preference was noted for polycyclic aromatic hydrocarbons with four to five benzene rings arranged in a nonlinear fashion, suggesting the stereochemical requirements of the protein binding site. The stability of the noncovalent interaction between the proteins and 3-methylcholanthrene was in the range of pH 7 to 9.