Low pH Buffer Research Articles

Capillary Electrophoresis of Insulin-like Growth Factors: Enhanced Ultraviolet Detection Using Dynamically Coated Capillaries and On-Line Solid-Phase Extraction

Insulin-like growth factor-I and -II (IGF-I and IGF-II) are difficult to separate and measure as a result of their homology, both structurally and immunologically. A number of binding proteins (BPs) which interact with the IGFs with high affinity complicate the ability to measure the IGFs accurately and reproducibly. Current methodology for measuring IGF is immuno-based and involves dissociation from the IGFs and removal of the binding proteins through sample acidification and removal by solid-phase adsorption. However, the net result is an assay that is time-consuming and, at best, semiquantitative. In an attempt to improve the reproducibility and accuracy of IGF-I and -II measurement, electrophoretic systems employing dynamically coated and bare silica capillaries were evaluated. Separations in bare silica capillaries in the presence or absence of the cationic additive, decamethonium bromide were ineffective for resolving IGF-I and IGF-II. However, when the capillary was coated dynamically with polybrene, IGF-I and -II could be resolved in a BSA sample matrix using a low pH buffer. Despite the fact that the IGFs could be resolved in the presence of an IGF-I analog used as an internal standard, polybrene recoating was required after as few as 12 runs and poor coating-to-coating reproducibility was observed. Use of polydiallyldimethylammonium chloride (PDMAC) as a dynamic cationic coating and a low pH buffer containing 0.5% PDMAC was found to be much more effective, providing reproducible separation of IGF-I and -II. It was found that PDMAC need not be included in the separation buffer to obtain reproducible analyses regarding IGF separation. Subsequently, functionality remained intact for as many as 35–40 consecutive analyses before recoating was required. Without the need for PDMAC in the buffer, on-line solid-phase extraction–capillary electrophoresis could be accomplished for detection of IGF-I and -II at concentrations as low 195 ng/ml.

High-pressure inactivation and sublethal injury of pressure-resistant Escherichia coli mutants in fruit juices.

The potential of high-pressure-resistant mutants of Escherichia coli to survive high-pressure pasteurization in fruit juices and in low-pH buffers was investigated. Treatments with up to 500 MPa of pressure caused only a limited direct inactivation of the mutants but resulted in an accelerated low-pH inactivation during subsequent storage.

Cationic proteins of neutrophil azurophilic granules: protein-protein interaction and blockade of NADPH oxidase activation.

We have previously reported inhibition of cell-free activation of the neutrophil superoxide-generating NADPH oxidase by a soluble cationic protein of neutrophil granules and by low concentrations of human defensin. Subcellular fractionation carried out in the current study indicated that the inhibitory substance was derived from azurophilic granules, was released into the medium on cell stimulation, and was resistant to phenylmethylsulfonyl fluoride (PMSF). Phorbol ester was the most effective stimulus for the release of the blocking activity. The possibility was raised that granule protein(s) act in vivo as negative modulators of superoxide production. Gel filtration of granule extract revealed a markedly retarded protein peak exhibiting oxidase-blocking activity and containing lysozyme as the main protein. Because lysozyme did not exert inhibitory effects on oxidase activation, association of the inhibitory protein with lysozyme was assumed. Indeed a column of immobilized lysozyme retained a fraction of the granule extract's oxidase-blocking activity. Elution with a low-pH buffer recovered a component capable of inhibition of the NADPH oxidase in stimulated neutrophils and in the cell-free system. The main 29-kDa protein band in the eluted fraction was identified as proteinase 3, a serine protease of azurophilic granules. Enzymatically active as well as PMSF-blocked conventionally purified proteinase 3 interfered with phorbol myristate acetate-induced superoxide release. These findings support the hypothesis that exocytosed granule constituents may prevent excessive activation of the NADPH oxidase.

Separation of pharmaceutical bases from neutral and acidic components by capillary electrochromatography

Capillary electrochromatography (CEC) has proved to be applicable to the separation of neutral and acidic (chromatographed in their ion-suppressed mode) compounds but a major factor holding back its further development has been its perceived inability to analyse basic compounds. In this paper we demonstrate that the addition of a competing base, such as triethylamine (TEA) or triethanolamine (TEOA), to a low pH buffer can achieve excellent CEC analysis of a range of strong pharmaceutical bases. Acceptable peak symmetry and efficiencies of up to 510000 plates m–1 were obtained for bases such as procainamide, nortriptyline and diphenhydramine on CEC Hypersil C18, C8 and phenyl reversed-phase packing materials. The effect of TEA or TEOA concentration and mobile phase pH on the separation behaviour of the bases is discussed. In addition, the paper reports the first example of the simultaneous CEC analysis of acids, bases and neutral compounds by using a mobile phase containing TEOA phosphate at pH 2.5.

Factors affecting soil acidification under legumes I. Effect of potassium supply

This study examined the effect of K (as K2SO4) supply on acid production under N2-fixing plants of lupin (Lupinus angustifolius L. cv. Gungurru) and clover (Trifolium subterraneum L. cv. Dalkeith) grown in a K-deficient soil with a low pH buffer capacity for 55 days in the glasshouse at 20/12 °C (day/night). Increasing K supply up to 240 mg K kg-1 soil markedly increased plant growth of both species but clover growth was more responsive than lupin. Growing plants for 55 days decreased soil pH by 0.65–0.85 units under lupin and 0.45–0.83 units under clover. The amounts of H+ produced per kg biomass (specific acid production) were the highest at the nil K supply, generally decreased with increasing K level up to 30 mg K kg-1 under lupin and up to 120 mg K kg-1 soil under clover and only slightly increased with further increasing K under lupin. Increasing K2SO4 supply proportionally increased plant uptake of K and SO 4 2- but generally decreased concentrations of Ca, Mg, Na, P and Cl. Specific acid production correlated well with concentrations of excess cations and ash alkalinity, and total acid production was strongly correlated with total excess cations and total ash alkalinity in plants. These relationships were not affected by K treatment and species. Specific acid production also correlated with plant Ca concentration but not with K concentration. In addition, lupin and clover extruded similar amounts of H+ per kg biomass produced. It is suggested that application of K2SO4 does not have a significant impact on acid production by lupin and clover.

Phosphorylation of the N-Formyl Peptide Receptor Is Required for Receptor Internalization but Not Chemotaxis

The human N-formyl peptide receptor (FPR) is a member of the family of leukocyte, G protein-coupled, chemoattractant receptors. To determine the role(s) of receptor phosphorylation in FPR processing and formylmethionylleucylphenylalanine (fMLF)-mediated chemotaxis, we utilized U937 cells expressing the recombinant wild type receptor and a mutant form of the FPR. This mutant, which lacks all of the serine and threonine residues in the C terminus of the receptor, DeltaST, has recently been shown to produce a receptor capable of fMLF binding and G protein activation but was demonstrated not to undergo fMLF-dependent phosphorylation or desensitization of the calcium mobilization response upon repeated exposure to agonist (Prossnitz, E. R. (1997) J. Biol. Chem. 272, 15213-15219). In this report, we examined the role of receptor phosphorylation in FPR internalization and leukocyte chemotaxis. Whereas the wild type receptor was rapidly internalized upon stimulation, the phosphorylation-deficient mutant was not, remaining entirely on the cell surface. In addition, contrary to the hypothesis that receptor processing and recycling are required for chemotaxis, we found no defect in the ability of the mutant FPR to migrate up a concentration gradient of fMLF. These results indicate that phosphorylation of the FPR is a necessary step in receptor internalization but that receptor phosphorylation, desensitization, and internalization are not required for chemotaxis.

Achievement of enantioselectivity of non-polar primary amines by a non-chiral crown ether

A non-chiral crown ether (18-crown-6), as a molecular modifier, along with cyclodextrin (CD) was used to achieve or enhance enantioselective separations of non-polar primary amines in capillary electrophoresis. Chiral separations of the primary amines are rarely achieved using cyclodextrins alone in capillary electrophoresis, because the three-point intermolecular interaction between the chiral amine and the cyclodextrin is not selective enough to yield resolution of the enantiomers of interest. To resolve these types of compounds, the non-chiral crown ether (18-crown-6) has been used with cyclodextrins (CDs) in the buffer. In the method, the amino group of the compounds is protonated using a low pH buffer solution and locked into 18-crown-6 ring forming a host-guest complex (amine+18-crown-6). The hydrophobic portion of the complex is then incorporated into the cavity of the cyclodextrin forming a secondary sandwich complex (18-crown-6+ amine+CD), in which the chiral center of the amine molecule is recognized. It is postulated that the sandwich complex results in a more rigid structure around the chiral center and more selective interaction between the chiral amine and cyclodextrin. This method can be used to separate a variety of non-polar primary amines, even when the chiral center is three-carbon atoms or more from the hydrophobic group.

Pyranose 2-oxidase from Phanerochaete chrysosporium--further biochemical characterisation.

Pyranose 2-oxidase (P2O) was purified 43-fold to apparent homogeneity from the basidiomycete Phanerochaete chrysosporium using liquid chromatography on phenyl Sepharose, Mono Q (twice) and phenyl Superose. The native enzyme has a molecular mass of about 250 kDa (based on native PAGE) and is composed of four identical subunits of 65 kDa. It contains three isoforms of isoelectric point (pI) 5.0, 5.05 and 5.15 and does not appear to be a glycoprotein. P2O is optimally stable at pH 8.0 and up to 60 degrees C. It is active over a broad pH range (5.0-9.0) with maximum activity at pH 8.0-8.5 and at 55 degrees C, and a broad substrate specificity. D-Glucose is the preferred substrate, but 1-beta-aurothioglucose, 6-deoxy-D-glucose, L-sorbose, D-xylose, 5-thioglucose, D-glucono-1,5-lactone, maltose and 2-deoxy-D-glucose are also oxidised at relatively high rates. A Ping Pong Bi Bi mechanism was demonstrated for the P2O reaction at pH 8.0, with a catalytic constant (kcat) of 111.0 s-1 and an affinity constant (Km) of 1.43 mM for D-glucose and 83.2 microM for oxygen. Whereas the steady-state kinetics for glucose oxidation were unaffected by the medium at pH > or = 7.0, at low pH both pH and buffer composition affected the P2O kinetics with the kcat/K(m) value decreasing with decreasing pH. The greatest effect was observed in acetate buffer (0.1 M, pH 4.5), where the kcat decreased to 60.9 s-1 and the K(m) increased to 240 mM. The activity of P2O was completely inhibited by 10 mM HgCl2, AgNO3 and ZnCl2, and 50% by lead acetate, CuCl2 and MnCl2.

Determination of histamine by capillary zone electrophoresis using a low-pH phosphate buffer: application in the analysis of fish and marine products

Histamine levels in samples of fish and marine products were determined by capillary zone electrophoresis using phosphate buffer at pH 2.44 and UV detection at 214 nm. A plot of the area of the peaks versus histamine concentration was linear over the range of 1–100 ppm with a correlation coefficient of 0.9996. Calibration using the height of the peak also gave good correlation, with a correlation coefficient of 0.9969, at concentrations between 1 ppm and 20 ppm. The recovery of added histamine in different samples of fish and fish products was, on average, 99.65%. The method described in this work was used for the quantitation of histamine in fish and fish products.

SSCP analysis of long DNA fragments in low pH gel.

Sensitivity of single-strand conformation polymorphism analysis of PCR products (PCR-SSCP analysis) is known to be decreased when the DNA fragments are longer than 300 bp. We examined effects of buffer ions in an attempt to extend the length limit of the analysis. It has been noted that addition of glycerol to the gel containing Tris-borate buffer enhances the sensitivity, but the effects of glycerol have been left unexplained. We found that the effects of glycerol are caused by the reduction of pH of the buffer by the reaction of glycerol and borate ion. We further extended these observations and found that sensitivity of SSCP can be greatly improved by running the electrophoresis in low pH buffer systems. Using a new buffer system and running the electrophoresis at a fixed temperature, we detected 27 of 31 known mutations of factor IX gene in six different sequence contexts ranging in length from 300 to 800 bp.

Preparation of Biotinylated, Affinity-Purified Antibodies for Enzyme-Linked Immunoassays Using Blotting Membrane as an Antigen Support

A method combining the affinity purification and biotinylation of antibodies has been developed utilizing the high-capacity binding of blotting membranes and glutaraldehyde treatment to immobilize antigen. Following reaction of the antisera with the membrane-bound antigen, the biotinylation of the antibodies is performedin situ,before release of the IgG–biotin complex with low pH buffer. The antibody is able to recognize antigen in subsequent reactions in enzyme-linked immunosorbent assays, and preliminary results have shown that the antibody can also be used in immunocytochemical localization of antigen in frozen sections. Biotinylationin situmay protect the variable region of the IgG compared to antibodies biotinylated in solution, thus increasing their antigen recognition. The method has the advantage of being suitable for multiple use of the antigen-coated membrane, making it particularly attractive where only small amounts of antigen may be available.

Experimental Investigation and Modeling of Uranium (VI) Transport Under Variable Chemical Conditions

The transport of adsorbing and complexing metal ions in porous media was investigated with a series of batch and column experiments and with reactive solute transport modeling. Pulses of solutions containing U(VI) were pumped through columns filled with quartz grains, and the breakthrough of U(VI) was studied as a function of variable solution composition (pH, total U(VI) concentration, total fluoride concentration, and pH‐buffering capacity). Decreasing pH and the formation of nonadsorbing aqueous complexes with fluoride increased U(VI) mobility. A transport simulation with surface complexation model (SCM) parameters estimated from batch experiments was able to predict U(VI) retardation in the column experiments within 30%. SCM parameters were also estimated directly from transport data, using the results of three column experiments collected at different pH and U(VI) pulse concentrations. SCM formulations of varying complexity (multiple surface types and reaction stoichiometries) were tested to examine the trade‐off between model simplicity and goodness of fit to breakthrough. A two‐site model (weak‐ and strong‐binding sites) with three surface complexation reactions fit these transport data well. With this reaction set the model was able to predict (1) the effects of fluoride complexation on U(VI) retardation at two different pH values and (2) the effects of temporal variability of pH on U(VI) transport caused by low pH buffering. The results illustrate the utility of the SCM approach in modeling the transport of adsorbing inorganic solutes under variable chemical conditions.

Binding of hemoglobin to the envelope of Porphyromonas gingivalis and isolation of the hemoglobin-binding protein.

The binding activity of the Porphyromonas gingivalis envelope and hemoglobin was examined over a wide range of pH values from 4.5 to 9.0. The binding activity in low-pH buffers was much higher than that at high pH; the optimum pHs for the binding were found to be 4.5 and 5.0. Since the hemoglobin bound to the envelope was found to dissociate in the pH 8.5 and 9.0 buffers, the binding is reversible. We hypothesized that hemoglobin-binding protein (HbBP), responsible for the binding to hemoglobin, exists in the envelope and confirmed its presence by dot blot determination with peroxidase-conjugated hemoglobin. Then we attempted to isolate HbBP from the solubilized (by a detergent) materials of the envelope by affinity chromatography. The molecular mass of HbBP was 19 kDa, and the isoelectric point was 4.3.

Immunoaffinity chromatography.

This unit describes the isolation of soluble or membrane-bound protein antigens from cells or homogenized tissue by immunoaffinity chromatography. The technique involves the elution of a single protein from an immunoaffinity column after prior elution of nonspecifically adsorbed proteins. To elute the bound antigen from the immunoaffinity matrix, the antibody-antigen interaction is destabilized by brief exposure to high-pH or low-pH buffer. The use of batch purification of antigens is an alternate procedure and results in shorter column loading times. The detergent octyl beta-D-glucoside can be used instead of Triton X-100 for elution. Because octyl beta-D-glucoside has a high critical micelle concentration (CMC), it can be readily removed by dialysis, as described.

Role of capillary electrophoresis methods in the drug development process

In the past several years, capillary electrophoresis (CE) has generated considerable interest from pharmaceutical companies for control of both the chiral and achiral purity of bulk drugs and drug products. This paper evaluates the use of CE as: (1) a technique complementary to HPLC for the determination of peak homogeneity of a drug, (2) for determination of chiral purity, and (3) for determination of achiral purity. It would be greatly advantageous if CE could be used to determine both the chiral and achiral purity in a single assay. This investigation compares the results obtained for the separation of the enantiomers of duloxetine using several neutral cyclodextrins to those obtained using anionic cyclodextrins (sulfobutyl ether derivatives) as chiral selectors added to the separation buffer. In addition, it reports chiral separations obtained by using neutral cyclodextrins in a sulfonic acid-coated capillary column, which give a negatively charged capillary surface and electro-osmotic flow even in low pH buffers. The possible mechanism of separation is discussed. © 1996 Wiley-Liss, Inc.

Capillary zone electrophoretic resolution of recombinant human bone morphogenetic protein 2 glycoforms an investigation into the separation mechanisms for an exquisite separation

Recombinant human bone morphogenetic protein 2 (rhBMP-2) is a disulfide-linked homodimeric glycoprotein ( M r = 30 000) which induces bone formation in vivo in several animal model systems. In this paper, we report the separation of a homogeneous rhBMP-2 sample into nine peaks by capillary zone electrophoresis (CZE), using a simple, pH 2.5, phosphate buffer containing no additives. The nine peaks have been identified to be glycoforms of rhBMP-2 [designated as (rhBMP-2) 2-(GlcNAc) 4(Man Z), where Z varies from 10 to 18]. The difference between any adjacent pair of peaks is only one mannose residue ( M r = 162). The ability of CZE to resolve rhBMP-2 glycoforms having the same charge and differing only 0.5% in molecular mass, without resorting to chemical complexation, is both unexpected and intriguing. Possible mechanisms explaining how the additional mannose can affect the mobility of rhBMP-2 glycoforms were explored. Zeta potentials of various glycoforms were calculated from their mobilities and interpreted in light of diffuse double layer parameters. Our results suggest that CZE employing a low-pH buffer, where proteins are highly charged, may be uniquely suitable for complex protein glycoform analysis.

Chiral Separation and Detection of Terbutaline and Ephedrine by Capillary Electrophoresis Coupled with Ion Spray Mass Spectrometry

The enantiomers of both terbutaline and ephedrine were separated by capillary electrophoresis using electrolyte solutions of heptakis(2,6-di-O-methyl)-beta-cyclodextrin in low pH buffers. The analytes were detected by ion spray mass spectrometry using selected ion monitoring or by selected reaction monitoring in the positive ion mode. Both the free drug enantiomers and the noncovalent enantiomer-cyclodextrin inclusion complexes for terbutaline can be detected simultaneously using this method. The feasibility of using CE/MS for chiral purity determination is demonstrated. In addition, a comparison of UV detection versus mass spectrometry detection for a spiked urine sample is included to demonstrate the selectivity and sensitivity advantages of the mass spectrometer as a CE detector.

A neural network, based on bicarbonate monitoring, to control anaerobic digestion

The use of a neural network simulation for monitoring and controlling an anaerobic digestion process by utilising data from an on-line bicarbonate alkalinity (BA) sensor is presented. The results collected from a series of induced disturbances, in the form of an increase in influent concentration, suggest that the instrument is very sensitive to BA changes and that the neural network is capable of rapid recognition of these disturbances. A scheme for the use of these changes is suggested that would allow on-line control of the digestion process in cases where the wastewater has a low pH buffering capacity.

Calorimetric detection of influenza virus induced membrane fusion

Membrane fusion induced by the hemagglutinin glycoprotein of influenza virus has been extensively characterized, but the mechanism whereby the protein achieves the merger of the viral and target membrane lipids remains enigmatic. Various lipid intermediate structures have been proposed, and the energies required for their formation predicted. Here, we have analyzed the enthalpies of fusion of influenza with liposomes by titration calorimetry. If a small sample of virus in a weak neutral pH buffer was added to an excess of liposomes at low pH, a two-component reaction was seen, composed of an exothermic reaction and a slower endothermic reaction. The exothermic reaction was the result of acid-base reactions between the neutral pH virus sample and low pH buffer and low-pH-induced changes in the virus. The endothermic reaction was not observed in the absence of liposomes and much reduced if acid-inactivated virus, which had lost its fusion but not its binding activity, was added to liposomes. The endothermic reaction was more temperature dependent than the exothermic reaction; its pH dependence corresponded with that of fusion and its enthalpy was higher if fusion was more extensive. These data indicate that most of the endothermic reaction was due to membrane fusion. The experimentally determined enthalpy of fusion, 0.6-0.7 kcal per mol of viral phospholipids, is much higher than expected on the basis of current theories about the formation of lipid intermediates during membrane fusion.

Proton inhibition of [ 3H]resiniferatoxin binding to vanilloid (capsaicin) receptors in rat spinal cord

Protons and capsaicin activate overlapping subsets of sensory nerves by opening ion conductances of similar properties. We have used the [ 3H]resiniferatoxin binding assay utilizing rat spinal cord membranes to elucidate the possible interaction of protons at the vanilloid (capsaicin) receptor. Using low pH (pH 6.0 and pH 5.0) buffers, a time-dependent gradual decrease was observed in specific resiniferatoxin binding. Protons inhibited resiniferatoxin binding with an IC 50 of pH 5.3 ± 0.1. In experiments in which the concentration of [ 3H]resiniferatoxin was varied, protons reduced the max value by approximately 40% with a corresponding 2-fold decrease in affinity. No change however, was observed in binding cooperativity (the Hill coefficients were 1.7 ± 0.1 and 1.6 ± 0.2 in the presence of pH 7.4 and pH 5.0 buffers, respectively). These changes in binding parameters are consist with a non-competitive or, alternatively, mixed inhibitory mechanism. The remaining resiniferatoxin binding sites bound capsaicin with an affinity ( K i=5.0 ± 1.0 μM) very similar to that determined in the presence of a pH 7.4 buffer ( K i = 3.0 ± 1.5 μM). A cyclooxygenase inhibitor, indomethacin (up to 10 μM), did not prevent the action of protons on resiniferatoxin binding; neither was it mimicked by prostanoids (prostaglandin I 2 and E 1, both at 100 μM). We conclude that protons interact at vanilloid receptors in the rat spinal cord; this interaction is either non-competitive or mixed in nature, and probably is not related to prostanoid generation. Protons and/or putative proton-generated mediators might represent endogenous modulators of the vanilloid receptor.