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F E Regnier

Publications and source records attributed to F E Regnier.

At least 91 records · Page 5Linked to original sources
PubMed1988

Synthesis of a non-porous, polystyrene-based strong anion-exchange packing material and its application to fast high-performance liquid chromatography of proteins.

Adsorbed coating technology has been used to produce a strong anion-exchange stationary phase on 3-micron non-porous poly(styrene-divinylbenzene) particles. In order to take full advantage of the excellent kinetic properties of the resultant packing material, small columns of 5 mm X 6 mm I.D. were used. These columns were pressure- and pH-stable and allowed protein separations to be made in less than 1 min at ambient temperature.

Chemical Phenomena↗
PubMed1988

Molecular orientation of immunoglobulin G at high concentration on an ion-exchange sorbent.

The change of molecular orientation of IgG, bound on a strong-anion-exchange surface, was studied using a generalization of the stoichiometric displacement model, over the entire range of protein adsorption isotherms. The Z number was found to decrease with increasing stationary phase protein concentration, approaching a limiting value. The analogy of the multiple equilibria model within highly cooperative identical binding sites was suggested as a possible way to evaluate the observed change in Z number with the protein concentration.

Adsorption↗
PubMed1988

Chromatography of proteins on hydrophobic interaction and ion-exchange chromatographic matrices: mobile phase contributions to selectivity.

The effect of mobile phase pH on the retention characteristics of eleven proteins was examined in hydrophobic interaction chromatography (HIC) on a SynChropak propyl stationary phase. Selectivity was shown to change with eluent pH. The effect of the displacing salt on the separation of proteins on a weakly hydrophobic weak-anion-exchange chromatography (AEC) packing was examined. Some differences in selectivity were observed when sodium sulfate was used as the displacing salt, compared to that observed with sodium chloride in the eluent. It was demonstrated that these AEC packings exhibited both electrostatic and hydrophobic properties, depending upon the type and concentration of salt used in the mobile phase. The addition of 20% ethylene glycol to the mobile phase was shown to reduce the hydrophobic interactions. The application of weakly hydrophobic weak-cation-exchange packings to HIC of proteins was demonstrated. Elution of such columns with descending sodium sulfate gradients was found to provide a selectivity different from that observed with a propyl stationary phase. Manipulation of mobile phases was shown to provide useful selectivity as a result of the combination of electrostatic and hydrophobic contributions to the separation process.

Chemical Phenomena↗
PubMed1988

Surface-mediated retention effects of subtilisin site-specific variants in cation-exchange chromatography.

Wild-type subtilisin and several site-specific variants were resolved on a strong cation-exchange column by isocratic elution and a series of sodium chloride concentrations. Changes in primary sequence at the protein surface have an observable effect on the chromatographic behavior of subtilisin. This supports the concept that three-dimensional structure determines which biopolymer surface residues are in position to interact with the stationary phase surface. The retention data fit the stoichiometric displacement model (SDM) of retention. Plots of ln k' vs. ln 1/[NaCl] yield values for the average number of ionic groups (Z) on the protein that interact with the support matrix. Application of the SDM to the chromatographic retention of the variants has uncovered an unusual phenomenon at the protein surface at low ionic strength. A SDM plot normally provides a linear relationship between ln k' and ln 1/[NaCl] with the slope corresponding to the Z number. This study revealed two lines differing in slope and intercept, indicating that the Z number of subtilisin changes at some intermediate ionic strength of the eluent. These results are attributed to some salt-induced protein surface event that triggers a change in structure. Chromatographic detection of this occurrence reflects the connection between the surface-mediated event and mobile phase ionic strength.

Amino Acids↗
PubMed1988

Chromatographic resolution of lysozyme variants.

There are seven avian lysozyme variants of nearly identical three-dimensional structure which have amino acid substitutions broadly distributed on their surface. By using these protein variants, it was possible to study the relationship between protein structure and chromatographic retention. It was determined that according to the mode of separation various regions of the proteins surface determine chromatographic retention. At one extreme, immunosorbents targeted a very small region on the protein surface. Hydrophobic interaction chromatography was an intermediate case in which one surface domain of the lysozymes controlled chromatographic behavior. At the opposite extreme, cation-exchange columns probed most of the protein surface. It was concluded that identification of random variations in protein structure will be most successfully detected by a separation mode that broadly targets the surface of a protein.

Animals↗
PubMed1988

Correlation of calcitonin structure with chromatographic retention in high-performance liquid chromatography.

Chromatographic selectivity and position specificity were examined by using deletion and substitution analogues of calcitonin (CT), a 32-amino acid polypeptide. The biological activity of CT has been shown to be related to structural features, including a regular spacing of hydrophobic and hydrophilic residues in positions 8-22. The effect of structure on retention behavior in hydrophobic interaction chromatography (HIC) and reversed-phase chromatography (RPC) of 19 CT analogues was examined. No simple correlation was found between chromatographic retention and amino acid composition. Deletion of one leucine residue reduced retention in both chromatographic systems, but the magnitude of the change depended upon the site at which the deletion occurred. For example, deletion analogues des(Leu16)-, des-(Leu12)-, des(Leu10)-, des(Leu9)-, des(Leu4)-, and salmon calcitonin had retention times of 5.22, 8.44, 11.64, 13.53, 15.45, and 20.28 min, respectively, in HIC in contrast to RPC retentions of 15.19, 15.84, 28.53, 27.57, 25.92 and 34.79 min, respectively. Serine deletion was also shown to be position-specific. Non-amphiphilic analogues were eluted before amphiphilic ones. Circular dichroic spectral studies showed that the CT analogues possessed little secondary structure in the HIC solvents in contrast to alpha-helix formation in RPC solvents. The HIC data provided indirect evidence of secondary structure, induced in the amphiphilic CT analogues at the HIC solvent-chromatographic surface interface. Solute-solvent interactions contributed to differences observed between the selectivity of RPC and HIC.

Amino Acid Sequence↗
PubMed1987

Effects of large sample loads on column lifetime in preparative-scale liquid chromatography.

In preparative-scale liquid chromatography of proteins, the use of high sample concentration and large sample mass may result in irreversible adsorption to the support surface. This can change the stationary phase characteristics, reduce the capacity, shorten the column lifetime and diminish the economic viability of a particular separation method. Column recycling and regeneration can influence the throughput (mass purified per time unit) and selectivity, and affect the reproducibility. The effects of large sample loads on column lifetime and performance were evaluated for three strong anion-exchange columns: (1) a silica support with a quaternized polyethyleneimine (PEI) coating, (2) a polymeric support with an adsorbed PEI coating which also was quaternized, and (3) a polymeric support with a proprietary quaternary amine stationary phase. The column capacity for proteins was measured by frontal chromatography and monitored as a function of cycle number. The column lifetime was determined by examining chromatographic properties subsequent to the frontal chromatography. The change in protein binding capacity was then compared to the change in nitrate binding capacity. The column performance was evaluated under analytical conditions by measuring the change in resolution of standard protein mixtures.

Albumins↗
PubMed1987

Selective non-adsorption preparative chromatography of bovine immunoglobulin Gl.

Bovine immunoglobulin Gl (IgGl) was purified from blood serum by isocratic elution through a mixed-bed ion-exchange column at low ionic strength. Selection of a mobile phase pH near the isoelectric point of IgG precluded its binding to the strong cation- and anion-exchange sorbents and allowed IgGl to be eluted isocratically. Under these conditions most of the other components in the serum sample were retained on the mixed-bed column. This approach gives excellent throughput and purity.

Adsorption↗
PubMed1987

The role of protein structure in chromatographic behavior.

Chromatographic retention is determined by a relatively small number of amino acids located in a chromatographic contact region on the surface of a polypeptide. This region is determined by the mode of separation and the amino acid distribution within the polypeptide. The contact area may be as small as a few hundred square angstroms in bioaffinity chromatography. In contrast, the contact region in ion exchange, reversed phase, hydrophobic interaction and the other nonbioaffinity separation modes is much broader, ranging from one side to the whole external surface of a polypeptide. Furthermore, structural changes that alter the chromatographic contact region will alter chromatographic properties. Thus, although immunosorbents can be very useful in purifying proteins of similar primary structure, they will be ineffective in discriminating between small, random variations within a structure. Nonbioaffinity columns complement affinity columns in probing a much larger portion of solute surface and being able to discriminate between protein variants.

Adsorption↗
PubMed1987

Retention mechanism of lactate dehydrogenase in anion-exchange chromatography.

The anion-exchange retention mechanism of lactate dehydrogenase (LDH) isoenzymes was examined under conditions where the muscle (M) subunit was not retained by the sorbent matrix. Using the stoichiometric displacement model of retention to quantitate the number (Zn) of ionic groups on the protein that interact with the sorbent matrix, it was determined that Zn for the LDH isoenzymes increases incrementally as the number of heart (H) subunits is increased up to a total of three H subunits. Both the MH3 and H4 isoenzymes have the same Zn. As the subunits in this tetrameric enzyme are arranged in a tetrahedral structure, these data indicate that steric limitations prevent any more than three subunits from interacting simultaneously with the sorbent surface. The reason why the MH3 and H4 isoenzymes could be separated is apparently that there are multiple equivalent faces in the H4 isoenzyme, and this both increases the probability and rate of adsorption and decreases the probability and rate of desorption for the H4 relative to the MH3 isoenzyme. It was concluded from these studies that the three-dimensional structure of a biopolymer will determine those surface residues which are in a position to interact with a sorbent surface and that multiple subunits or domains of a protein may act cooperatively over considerable distances in biopolymer-surface interactions.

Chromatography, Ion Exchange↗
PubMed1987

Introduction of 5'-terminal functional groups into synthetic oligonucleotides for selective immobilization.

Oligodeoxyribonucleotides terminating in a 5'-primary amine group are synthesized using solid-phase supported phosphoramidite chemistry. The 5'-terminal amine group in the deprotected oligomers is further derivatized with either succinic anhydride to give 5'-carboxylic acid or with dithiobis(succinimidylpropionate) followed by treatment with dithioerythritol to produce 5'-thiol-terminated oligonucleotides. The 5'-thiol-terminated oligonucleotides are selectively immobilized on solid supports containing either p-chloromercuribenzoate or 2,2'-dithiobis(5-nitropyridine) activated thiol groups.

Amines↗
PubMed1987

Chromatography of complex protein mixtures.

This review has shown that a variety of chromatographic techniques are available for fractionating proteins. Fortunately, high-quality columns of every type described in this review are commercially available. Most water-soluble proteins may be eluted from size-exclusion, hydrophobic-interaction, ion-exchange, metal chelate, and bioaffinity columns with ease. When this is the case, high recovery and retention of biological activity are the norm. The exception is reversed-phase chromatography where the organic solvents and acids used in polypeptide elution denature many proteins. When problems do occur, they are generally the result of unique structural features of the protein. Very hydrophobic proteins have presented the biggest problem in that they are difficult to solubilize, particularly with retention of biological activity. It has been found that zwitterionic and non-ionic detergents are the most suitable solubilizing agents, but urea has also been used in cases where hydrophobic interacts are not as strong. Unfortunately, there is still an element of trial-and-error in selecting the most suitable solubilizing agent. Heterogeneous glycosylation of proteins also presents a problem. Both neutral and charged monosaccharides can be incorporated into proteins through multiple steps at several sites. Thus, there is the potential in a sample for a large number of glycoprotein species which have the same polypeptide backbone and differing amounts of oligosaccharide. A problem arises when size-exclusion, ion-exchange, hydrophobic-interaction, reversed-phase and bioaffinity systems begin to discriminate between these very similar glycoprotein species. Chromatographic peaks can become very broad, due to incomplete fractionation, and the polypeptide chain of interest can be associated with multiple peaks. The separation of glycoproteins requires much more study before logical procedures can be suggested for column selection and operation. Aggregated species are another class of proteins which present occasional problems. Multimeric proteins are adsorbed to sorbents by a series of forces, among which are hydrogen bonding, hydrophobic interactions, and electrostatic forces. These forces are also responsible for the maintenance of quaternary structure in proteins. When the same forces dominate both retention of protein structure and adsorption at the sorbent surface, the quaternary structure of the protein can be disrupted during elution. Very basic proteins also present a problem in some cases. Columns with residual negative charges, such as a silica-based reversed-phase column, adsorb anionic species so strongly that they are difficult to elute.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Synthesis and immobilization of a novel acridine derivative on microparticulate silica. A study of its interactions with single-stranded oligonucleotides by high-performance liquid chromatography.

A novel approach for immobilizing acridine on 5-microns silica gel is described. The acridine moiety is functionalized with a carboxylic acid group at its reactive 9-position and activated, leading to 9-acridinylpropionic acid N-hydroxysuccinimide ester. This derivative is efficiently bound to the silica matrix through a primary aliphatic amine group at the end of a fifteen-atom spacer arm. The chromatographic properties of the final stationary phases, as evaluated with d(T)10 and d(A)10 at various pH values and organic solvent concentrations, resemble those of hydrophobic weak anion exchangers. When a secondary amine group is placed close to the acridine moiety in one of the packings, enhanced binding of the oligodeoxyribonucleotides is observed that goes beyond a purely additive effect.

Acridines↗
PubMed1987

Poly(styrene-divinylbenzene)-based strong anion-exchange packing material for high-performance liquid chromatography of proteins.

An adsorbed polyethyleneimine coating was applied to sulfonated macroporous, microparticulate poly(styrene-divinylbenzene). The chemical, physical and chromatographic properties of the resulting strong anion-exchange packing material were thoroughly characterized. The dynamic load capacity of the experimental packing was comparable to that of large-pore diameter silica. Good recoveries of protein mass and enzyme activity were achieved. The new column withstood a variety of cleaning procedures and prolonged exposure to aqueous base. The retention times on the polystyrene-based column were similar to those on a silica and a commercial, polymeric, strong anion-exchange column. Chromatographic resolution of the new packing material was equal or superior to that provided by the other two packings.

Animals↗