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Biomedical subjects

F E Regnier

Publications and source records attributed to F E Regnier.

At least 109 records · Page 6Linked to original sources
PubMed1987

Characterization of osmotin : a thaumatin-like protein associated with osmotic adaptation in plant cells.

Cultured tobacco (Nicotiana tabacum var Wisconsin 38) cells adapted to grow under osmotic stress synthesize and accumulate a 26 kilodalton protein (osmotin) which can constitute as much as 12% of total cellular protein. In cells adapted to NaCl, osmotin occurs in two forms: an aqueous soluble form (osmotin-I) and a detergent soluble form (osmotin II) in the approximate ratio of 2:3. Osmotin-I has been purified to electrophoretic homogeneity, and osmotin-II has been purified to 90% electrophoretic homogeneity. The N-terminal amino acid sequences of osmotins I and II are identical through position 22. Osmotin-II appears to be much more resistant to proteolysis than osmotin-I. However, it cross-reacts with polyclonal antibodies raised in rabbits against osmotin-I. Osmotin strongly resembles the sweet protein thaumatin in its molecular weight, amino acid composition, N-terminal sequence, and the presence of a signal peptide on the precursor protein. Thaumatin does not cross-react with antiosmotin. An osmotin solution could not be detected as sweet at a concentration at least 100 times that of thaumatin which could be detected as sweet. Immunocytochemical detection of osmotin revealed that osmotin is concentrated in dense inclusion bodies within the vacuole. Although antiosmotin did not label organelles, cell walls, or membranes, osmotin appeared sparsely distributed in the cytoplasm.

Journal Article↗
PubMed1986

Factors contributing to intrinsic loading capacity in silica-based packing materials for preparative anion-exchange protein chromatography.

Properties of the matrix and stationary phase which affect the intrinsic loading capacity of silica-based packing materials for preparative anion-exchange chromatography of proteins were investigated. Polyethyleneimine-coated controlled porosity glass beads ranging from 100 to 2000 A in pore diameter were used to evaluate the effects of pore diameter and surface area. Protein binding was found to depend on accessible, rather than total, support surface area. Consequently, wide-pore, high surface area media provide maximum intrinsic loading capacity. Increasing the number of positively charged sites on the stationary phase by increased coating or by quaternization of amines increases hemoglobin-binding capacity.

Amines↗
PubMed1986

Multimodal liquid chromatography columns for the separation of proteins in either the anion-exchange or hydrophobic-interaction mode.

Several high-performance stationary phases suitable for protein chromatography were synthesized. Columns packed with these materials could be operated independently in either the anion-exchange or hydrophobic-interaction mode. Two approaches were used to prepare these materials. In the first method, a polyamine was adsorbed on the surface of macroporous silica and then crosslinked with a multifunctional oxirane. The hydrophobicity of the crosslinking agent and the extent of interconnection were used to modulate the electrostatic and solvophobic interactions. The second approach also utilized a crosslinked polyamine stationary phase; however, the forces of interaction were attenuated through controlled acylation of surface amines with a small anhydride molecule. The resolving ability of these columns, functioning in either mode, was comparable to commercial high-performance liquid chromatographic columns, designed to operate by a single retention mechanism. Column selectivity for proteins was completely different in each mode. Protein fractions collected from a multimodal column, operated in the anion-exchange mode, could be further purified by rechromatographing them on the same column in the hydrophobic-interaction mode. Utility of the multimodal column was demonstrated with the fractionation of several cytochromes and ferredoxins from the cyanobacterium Microcystis aeruginosa.

Chemical Phenomena↗
PubMed1986

Preparation and evaluation of inorganic anion-exchange sorbents not based on silica.

The use of alternate macroporous (greater than 200 A) inorganic support materials in the preparation of pellicular anion-exchange packings was explored. Alumina, magnesia, titania, and a zirconia-coated silica were chosen for comparison with silica. The stationary phase attached to the support surfaces was an adsorbed polyethyleneimine, crosslinked with 1,4-butanediol diglycidyl ether. Packing materials were characterized by static elemental analyses, chromatographic retention, static loading capacity and pH stability. Titania, alumina, and zirconyl-clad silica packings were found to be substantially more stable under alkaline conditions than silica-based materials. The data show that the stationary phase was successfully bonded in all cases and functioned in anion-exchange chromatography. When the surface area and pore diameter of these alternate materials is equivalent to silica, there is little difference in chromatographic properties.

Animals↗
PubMed1986

Solute and mobile phase contributions to retention in hydrophobic interaction chromatography of proteins.

Hydrophobic interaction chromatography utilizes high salt concentrations mobile phases to induce an interaction between a weakly hydrophobic matrix and exposed hydrophobic amino acids of a native protein. Proteins with a hydrophilic exterior have shorter retention times on a hydrophobic interaction column than do proteins with more hydrophobic exteriors. To examine the effect of amino acid substitutions on protein retention, lysozyme isolated from related bird species was chromatographed on a hydrophobic interaction column at increasing ammonium sulfate concentrations. Chromatographic retention deviated only when amino acid substitutions occurred on the surface of lysozyme opposite the catalytic cleft. This area may constitute a contact surface area and extends from Residue 41 to 102 and from 75 to the alpha-helical region starting with Residue 89. Retention was analyzed by plotting log k' versus the molal concentration of ammonium sulfate. The slope did not deviate significantly for each of the bird lysozymes, indicating a similar contact surface area. However, there was significant deviation in the intercept of each of the lysozyme lines, which probably reflects the strength of the hydrophobic interaction. The intercept increased as the lysozyme became more hydrophobic. Hydrophilic amino acid substitutions affected retention as much as hydrophobic ones. The ionization state of histidine residues within the contact area between lysozyme and the column surface also influenced retention. An uncharged histidine residue increased retention, while a decrease in retention was seen with a charged histidine residue. The amino acid substitutions did not appear to affect the size of the hydrophobic contact surface area, but rather the strength of the hydrophobic interaction. The effect of salt composition on protein retention indicated that factors other than surface tension could influence retention. These factors appear to include protein hydration and specific interactions between the protein and the salt ions. Of these, the latter may or may not result in an alteration in protein structure. The magnitude of the effect of salt composition was found to be dependent upon the protein.

Amino Acid Sequence↗
PubMed1986

Application of the stoichiometric displacement model of retention to anion-exchange chromatography of nucleic acids.

The stoichiometric displacement model has been refined in its application to anion-exchange chromatography. The revised stoichiometric displacement model has been shown to be valid for anion-exchange chromatography with respect to all particulars of the model tested. It has been shown that the use of displacing agent activity is not a necessary condition for valid application of the stoichiometric displacement model to anion-exchange chromatography. While the cation of the displacing salt can influence anion-exchange chromatography, the data indicate that the displacing anion is of primary importance. It has been shown that solutes with three-dimensional structure have Zn value to solute charge ratios less than unity, and that the stoichiometric displacement model may be useful as a probe of solute three-dimensional structure.

Adenine↗
PubMed1986

Synthesis of cation-exchange stationary phases using an adsorbed polymeric coating.

We have prepared several silica-based cation-exchange materials that were suitable for the high-performance liquid chromatography of basic proteins. Two synthetic routes were examined. Central to both procedures was the adsorption of a low molecular weight polyamine. One method crosslinks the adsorbed polyamine with a multifunctional oxirane, which is then extensively derivatized with a monomeric cyclic anhydride. The second involves an adsorbed uncrosslinked polyethyleneimine layer which is reacted with polyacrylic anhydride, thereby crosslinking and imparting anionic character simultaneously. The resulting media prepared by either of these methods bound more than 40 mg of hemoglobin per gram of support depending on the reaction conditions. These cation-exchange stationary phases also exhibited good chromatographic performance, successfully resolving (horse heart) cytochrome c and lysozyme. Two of the more promising support materials were effectively used to isolate cytochrome c553 from a crude extract of cyanobacteria.

Adsorption↗
PubMed1986

Synthesis of an adsorbed reversed-phase packing material for the separation of proteins and peptides.

We have described the preparation and chromatographic evaluation of an adsorbed hydrophobic stationary phase suitable for reversed-phase chromatography of proteins and peptides. The synthetic procedure involves three steps: the adsorption of a polyamine to the silica surface; crosslinking of the adsorbed polyamine layer with a bis-phenyl difunctional epoxide; and the benzoylation of the remaining accessible amino groups. Performance of this chromatographic material compared favorably with SynChropak RP-8 silica (SynChrom, Linden, IN, U.S.A.) and was stable to 40% formic acid. Good separations were obtained between the components of sample mixtures containing proteins or the cyanogen bromide fragments of sperm whale myoglobin. However, in both cases, the adsorbed hydrophobic stationary phase was less retentive. Furthermore, this medium exhibited slightly different selectivity. Whereas the heme which was present in the cyanogen bromide digest of myoglobin desorbed as the second peak from the RP-8 column, it eluted last from the adsorbed stationary phase. Comparable performance, acid stability and alternate selectivity suggest that this material is an interesting alternative to organosilane reversed-phase coatings.

Acids↗
PubMed1985

Stoichiometric displacement of solvent by non-polar solutes in reversed-phase liquid chromatography.

This paper proposes a retention model which predicts that the displacement of non-polar solutes from a reversed-phase chromatographic column is accompanied by the adsorption of a stoichiometric number (Z) of solvent molecules. The number of solvent molecules involved in this process is a function of both solute and solvent contact surface areas. Increasing solute contact surface area would increase Z whereas increasing solvent contact surface area would decrease the Z value for a specific solute. The experimental observations presented are consistent with this model. Further predictions of the model are that (1) plots of log k' versus the inverse log of solvent concentration will be non-linear at solvent concentrations where the surface of a reversed-phase support is not fully solvated, and (2) only a portion of the total non-polar surface area of a molecule actually contacts the surface of a reversed-phase support. Non-linearity in plots of log k' versus the inverse log of solvent concentration was in fact observed at solvent concentrations where solvation of the reversed-phase support is incomplete.

Benzene Derivatives↗
PubMed1985

High-performance anion-exchange chromatography of oligonucleotides.

Several types of high-performance silica-based supports have been found to be effective in the separation of polynucleotides. The principal difference in these materials is the type of bonded phase and the method by which it is attached to the silica support. One approach is the coupling of stationary-phase groups to the surface through siloxane bonding. This technique is simple and produces a material of high capacity and resolution, but it suffers from poor bonded-phase stability. An alternative approach is the adsorption of low-molecular-weight polyethylene imines (PEI) that are crosslinked into a surface film. The stationary phase is held in place by adsorption of the film at multiple sites. A previous report on this material showed the resolution of oligonucleotides containing up to 30 bases. This paper reports further optimization of the PEI bonding chemistry in the preparation of HP-IEC columns for oligonucleotides and tRNA species. Quaternization of the ion-exchange matrix was found to increase resolution of oligonucleotides from 30 to 50 bases. The same support was found to be capable of resolving multiple tRNA species. Separations were achieved on small (0.42 X 5 cm) columns, using a 60- to 120-min ammonium sulfate gradient. The initial solvent was 15% acetonitrile in 0.05 M potassium phosphate (pH 5.9). The addition of 1 M ammonium sulfate to the initial solvent was used to prepare the final solvent.

Adsorption↗
PubMed1985

Stationary phase contributions to retention in high-performance anion-exchange protein chromatography: ligand density and mixed mode effects.

Several anion-exchange stationary phases (based on polyethyleneimine-coated silica) were synthesized so as to vary in ligand density and hydrophobicity. These materials were first examined for hemoglobin-binding capacity and then evaluated chromatographically. Protein binding, retention and resolution increased concomitantly with ligand density. Ferritin (molecular weight 440,000) could not be eluted from the more highly-charged surfaces, but was desorbed from a low ligand density support. The above parameters also varied with the hydrophobic character of the stationary phase. Retention and resolution increased as more hydrophobic moieties were added. Data from a non-ionic hemoglobin-binding assay correlated reasonably well with anticipated matrix hydrophobicities. Possible explanations and applications of the observed phenomena are discussed.

Amines↗
PubMed1984

Comparison of hydrophobic-interaction and reversed-phase chromatography of proteins.

The variable hydrophobic nature of proteins allows their separation through differential hydrophobic surface interactions. From these observations two modes of protein chromatography have been developed, hydrophobic-interaction chromatography (HIC) and reversed-phase chromatography (RPC). Selectivity of the HIC column can be easily manipulated by changing mobile phase variables. Protein retention was increased by decreasing the pH from neutrality or by using a salt with a greater "salting-out" ability. In addition, selectivity can be altered through chemical modification of the matrix surface. Protein retention and resolution decreased concomitantly with matrix ligand density. There were several major differences in HIC and RPC selectivity. Hydrophilic proteins such as cytochrome c and myoglobin were weakly retained on the HIC column but strongly retained on the RPC column. In contrast, a hydrophobic protein such as beta-glucosidase was strongly retained on the HIC column and only weakly retained on the RPC column. Other proteins were retained equally by RPC and HIC columns. Load capacity on the HIC column was determined by plotting resolution as a function of protein load. Resolution decreased significantly after 7.5 mg of total protein had been loaded onto the column per cm3 of column material. Samples of lactic dehydrogenase and alpha-chymotrypsin ranging in size from 10-200 micrograms were recovered from an HIC column with greater than 86% enzymatic activity in all cases. The recovery of enzymatic activity of alpha-chymotrypsin ranged from 55-91%, while none of the activity of beta-glucosidase was recovered from the RPC column.

Animals↗
PubMed1984

Retention model for proteins in reversed-phase liquid chromatography.

This paper presents a retention model for proteins on an reversed-phase chromatography support in which retention is a function of the number (Z) of solvent molecules required to displace the solute from the surface. An equation is derived that relates the capacity factor of a protein to the displacing agent concentration and the stoichiometry of solvent-solute displacement. Experimental tests of the model indicate that each protein has a unique Z value and that Z is directly proportional to the molecular weight of a series of proteins when 60% formic acid is used as the mobile phase additive. This relationship is attributed to a direct relationship between Z and the contact surface area between polypeptide solutes and the support. Desorption curves for proteins also become more convex with increasingly molecular weight, as predicted by the retention model. In the solvent series of methanol, ethanol, propanol, the Z number decreases from the C1 to C3 alcohol. The Z number for any particular solvent is also related to other mobile phase additives, such as acids, and the concentration of additives.

Chemical Phenomena↗
PubMed1984

Adsorption of pepsin by aluminum hydroxide II: Pepsin inactivation.

Pepsin adsorbed on gibbsite or boehmite, non-acid-reactive forms of aluminum hydroxide, had a significantly lower activity than pepsin in solution. IR and desorbed pepsin activity studies showed that the reduced activity of adsorbed pepsin was not due to denaturation of pepsin on adsorption. Steric occlusion of the active site, following pepsin adsorption, was responsible for the lower activity of pepsin adsorbed on gibbsite. The porous morphology of boehmite caused diffusional resistance and steric exclusion, contributing to the decreased activity of adsorbed pepsin. The specific inactivation of pepsin by adsorption on aluminum hydroxide may be important in ulcer therapy.

Adsorption↗
PubMed1984

High-performance hydrophobic interaction chromatography of proteins.

A new, weakly hydrophobic, high-performance liquid chromatography column has been developed for the separation of native proteins based on their relative hydrophobicities. Starting with a covalently bound, hydrophilic polyamine matrix, packing materials were synthesized through acylation with anhydrides and acid chlorides of increasing chain length to obtain increasingly hydrophobic surfaces. Proteins in aqueous buffers were induced to bind hydrophobically to the columns by the use of high salt concentrations in the mobile phase. Elution was achieved by decreasing the ionic strength of the solvent in a linear gradient. A mixture of cytochrome c, conalbumin, and beta-glucosidase was used as a standard to test the resolving power of newly synthesized columns. On a 4-cm butyrate column, baseline resolution was achieved in 20 min with a gradient of 3.0 mu sodium sulfate in 0.1 M potassium phosphate buffer, pH 7.0, to water. The static loading capacity for each column was determined using a hemoglobin binding assay. Capacities normally ranged between 150 and 180 mg of hemoglobin per gram of support. Since proteins are not denatured in hydrophobic interaction chromatography, enzymes eluted from the column retained enzymatic activity. Samples of alpha-amylase and beta-glucosidase ranging in size from 10 to 200 micrograms were recovered from the butyrate column with greater than 92% enzymatic activity in all cases. In a single trial, the enzyme citrate synthase was recovered from the benzoate column with 92% retention of enzymatic activity.

Chemical Phenomena↗
PubMed1983

High-performance liquid chromatography of biopolymers.

The ability to separate biological macromolecules with good resolution on liquid chromatographic columns has depended on the development of suitable packing materials. In size exclusion chromatography, molecules are separated by size on the basis of differential permeation of the packing. Ion exchange, hydrophobic interaction (or reversed-phase), and affinity chromatography are all surface-mediated separation methods, although they depend on different retention mechanisms. High-performance liquid chromatographic columns designed for biopolymers offer major advantages over conventional columns in both speed and resolving power. The exponential growth of literature on the high-performance separation of peptides and proteins in particular indicates that the technique will become the dominant form of column liquid chromatography.

Chromatography, Affinity↗