D.R.K. Harding

Hydrophobic charge induction chromatography: salt independent protein adsorption and facile elution with aqueous buffers

A new form of protein chromatography, hydrophobic charge induction, is described. Matrices prepared by attachment of weak acid and base ligands were uncharged at absorption pH. At low ligand densities, protein adsorption was typically promoted with lyotropic salts. At higher ligand densities, chymosin, chymotrypsinogen and lysozyme were adsorbed independently of ionic strength. A pH change released the electrostatic potential of the matrix and weakened hydrophobic interactions, inducing elution. Matrix hydrophobicity and titration range could be matched to protein requirements by ligand choice and density. Both adsorption and elution could be carried out within the pH 5-9 range.

High-pressure liquid chromatography of peptides and proteins : II. The use of phosphoric acid in the analysis of under-ivatised peptides by reversed-phase high-pressure liquid chromatography

Abstract The chromatographic properties of a range of peptides varying in size from di- to decapeptide have been investigated by reversed-phase high-pressure liquid chromatography. A new set of conditions, namely, the addition of phosphoric acid to the mobile phase, has been found to have very real advantages in the analysis of underivatised peptides. These conditions allowed marked alterations in retention times, improvement in reproducibility and excellent resolution of peptides differing by as little as a single amino acid. A major advantage of phosphoric acid is that it can be used successfully in the range 195–220 nm which makes it compatible with the use of variable wavelength UV monitors as sensitive detectors in high-pressure liquid chromatography. In addition, the use of phosphoric acid permits the significant lowering of concentrations of organic solvents in the mobile phase, thus reducing the possibility of denaturation or precipitation.

Chemosensitization of Fluconazole Resistance in Saccharomyces cerevisiae and Pathogenic Fungi by a d-Octapeptide Derivative

ABSTRACT Hyperexpression of the Saccharomyces cerevisiae multidrug ATP-binding cassette (ABC) transporter Pdr5p was driven by the pdr1-3 mutation in the Pdr1p transcriptional regulator in a strain (AD/PDR5+) with deletions of five other ABC-type multidrug efflux pumps. The strain had high-level fluconazole (FLC) resistance (MIC, 600 μg ml−1), and plasma membrane fractions showed oligomycin-sensitive ATPase activity up to fivefold higher than that shown by fractions from an isogenic PDR5-null mutant (FLC MIC, 0.94 μg ml−1). In vitro inhibition of the Pdr5p ATPase activity and chemosensitization of cells to FLC allowed the systematic screening of a 1.8-million-member designer d-octapeptide combinatorial library for surface-active Pdr5p antagonists with modest toxicity against yeast cells. Library deconvolution identified the 4-methoxy-2,3,6-trimethylbenzensulfonyl-substituted d-octapeptide KN20 as a potent Pdr5p ATPase inhibitor (concentration of drug causing 50% inhibition of enzyme activity [IC50], 4 μM) which chemosensitized AD/PDR5+ to FLC, itraconazole, and ketoconazole. It also inhibited the ATPase activity of other ABC transporters, such as Candida albicans Cdr1p (IC50, 30 μM) and Cdr2p (IC50, 2 μM), and chemosensitized clinical isolates of pathogenic Candida species and S. cerevisiae strains that heterologously hyperexpressed either ABC-type multidrug efflux pumps, the C. albicans major facilitator superfamily-type drug transporter BenRp, or the FLC drug target lanosterol 14α-demethylase (Erg11p). Although KN20 also inhibited the S. cerevisiae plasma membrane proton pump Pma1p (IC50, 1 μM), the peptide concentrations required for chemosensitization made yeast cells permeable to rhodamine 6G. KN20 therefore appears to indirectly chemosensitize cells to FLC by a nonlethal permeabilization of the fungal plasma membrane.

Synthesis and characterisation of chemically modified chitosan microspheres

Abstract Chemically modified chitosan was synthesised by graft copolymerisation of poly(ethylene glycol) diacrylate macromonomer onto chitosan backbone. The polymerisation was initiated by ceric ammonium nitrate. Graft copolymers were prepared by varying the concentration of chitosan and the macromonomer. These graft copolymers were characterised by FT-IR, 1H-NMR and DSC techniques. Microspheres based on chitosan and polymer grafted chitosan were prepared by a polymer dispersion technique. Microspheres were characterised by scanning electron microscopy, (SEM) particle size distribution analysis and DSC methods. A comparative study in relation to the structural deviation among chitosan and modified chitosan microspheres is discussed. These chemially modified chitosans appeared to be hydrophilic and form aggregates during microsphere preparation.

One step purification of chymosin by mixed mode chromatography.

Mixed mode Sepharose and Perloza bead cellulose matrices were prepared using various chemistries. These matrices contained hydrophobic (aliphatic and/or aromatic) and ionic (carboxylate or alkylamine) groups. Hydrophobic amine ligands were attached to epichlorohydrin activated Sepharose (mixed mode amine matrices). Hexylamine, aminophenylpropanediol and phenylethylamine were the preferred ligands, on the basis of cost and performance. Other mixed mode matrices were produced by incomplete attachment (0-80%) of the same amine ligands to carboxylate matrices. The best results were obtained using unmodified or partially ligand-modified aminocaproic acid Sepharose and Perloza. High ligand densities were used, resulting in high capacity. Furthermore, chymosin was adsorbed at high and low ionic strengths, which reduced sample preparation requirements. Chymosin, essentially homogeneous by electrophoresis, was recovered by a small pH change. The methods described were simple, efficient, inexpensive and provided very good resolution of chymosin from a crude recombinant source. The carboxylate matrices had the best combination of capacity and regeneration properties. The performance of Sepharose and Perloza carboxylate matrices was similar, but higher capacities were found for the latter. Because it is cheaper and can be used at higher flow rates, Perloza should be better suited to large scale application. High capacity chymosin adsorption was found with carboxymethyl ion exchange matrices, but low ionic strength was essential for adsorption and the purity was inferior to that of the mixed mode matrices.

Salt-independent adsorption chromatography: new broad-spectrum affinity methods for protein capture

The role of chromatography in capture is reviewed in terms of the special requirements imposed by the processing of very crude feedstocks. Adsorption methods which are not significantly affected by variations of feedstock ionic strength are highlighted. Methods are compared in terms of simplicity, robustness, selectivity and ease of elution. The application of such methods to enzyme and antibody purifications is summarised. Particular emphasis is placed on high ligand density methods, which have potential for broad-spectrum application.

High-pressure liquid chromatography of peptides and proteins XI. The use of cationic reagents for the analysis of peptides by high-pressure liquid chromatography

This report describes the effect of different cationic reagents (tetraalkylammonium, alkylammonium and inorganic salts) on the retention times of di- to pentapeptides chromatographed on a reversed-phase support (i.e. a μBondapakalkylphenyl column). Several trends are apparent with these reagents which can be explained on the basis of either ion-pairing or ion-exchange interaction of the reagent with the peptide sample. Reagents which generate in solution small highly solvated cations, e.g. Li, Na or Mg salts, give retention times similar to those obtained for ammonium salts. Tetraethylammonium salts give a modest increase in retention times relative to ammonium salts. By contrast, hydrophobic cations with long or bulky carbon chains. e.g. tetrabutylammonium or dodecylammonium ions, cause substantial decreases in retention times, resulting in very rapid elution of all peptides examined from the reversed-phase column. These observations are consistent with the composite interplay of ion-pair partition and dynamic ion-exchange effects for the cationic reagents. The use of a mixture of dodecylammonium acetate and sodium dodecylsulphate for the analysis of peptides and proteins is described. It is anticipated that such a chromatographic system will he useful for the analysis of proteins which readily aggregate.

High-pressure liquid chromatography of peptides and proteins : VI. Rapid analysis of peptides by high-pressure liquid chromatography with hydrophobic ion-pairing of amino groups

Abstract This report describes the use of hydrophobic ion-pairing reagents in the rapid analysis of peptides by reversed-phase high-pressure liquid chromatography. It was found that combination of a hydrophobic anion such as hexanesulphonate with the cationic groups (R P + of a peptide resulted in a decreased polarity of the sample. This change in polarity resulted in an increased retention time on a μBondapak-alkylphenyl column. In addition, the use of the different ion-pairing reagents allowed dramatic changes in the selectivity of the reversed-phase system. This is demonstrated with peptides which range from tri- to heptapeptides using the following ion-pairing reagents: phosphoric acid, sodium hexanesulphonate and sodium dodecycl sulphate.

Surface-Active Fungicidal d-Peptide Inhibitors of the Plasma Membrane Proton Pump That Block Azole Resistance

ABSTRACT A 1.8-million-member d-octapeptide combinatorial library was constructed in which each member comprised a diversity-containing N-terminal pentapeptide and a C-terminal amidated triarginine motif. The C-terminal motif concentrated the library members at the fungal cell surface. A primary screen for inhibitors of Saccharomyces cerevisiae and Candida albicans growth, together with an in vitro secondary screen with the S. cerevisiae plasma membrane ATPase (Pma1p) as a target, identified the antifungal d-octapeptide BM0 (d-NH2-RFWWFRRR-CONH2). Optimization of BM0 led to the construction of BM2 (d-NH2-RRRFWWFRRR-CONH2), which had broad-spectrum fungicidal activity against S. cerevisiae, Candida species, and Cryptococcus neoformans; bound strongly to the surfaces of fungal cells; inhibited the physiological activity of Pma1p; and appeared to target Pma1p, with 50% inhibitory concentrations in the range of 0.5 to 2.5 μM. At sub-MICs (<5 μM), BM2 chemosensitized to fluconazole (FLC) S. cerevisiae strains functionally hyperexpressing fungal lanosterol 14α-demethylase and resistance-conferring transporters of azole drugs. BM2 chemosensitized to FLC some FLC-resistant clinical isolates of C. albicans and C. dubliniensis and chemosensitized to itraconazole clinical isolates of C. krusei that are intrinsically resistant to FLC. The growth-inhibitory concentrations of BM2 did not cause fungal cell permeabilization, significant hemolysis of red blood cells, or the death of cultured HEp-2 epithelial cells. BM2 represents a novel class of broad-spectrum, surface-active, Pma1p-targeting fungicides which increases the potencies of azole drugs and circumvents azole resistance.

High-density ligand attachment to brominated allyl matrices and application to mixed mode chromatography of chymosin

Abstract Allylated cellulose, agarose and methacrylate matrices, activated with allyl bromide or allyl glycidyl ether, were modified by aqueous bromination, preferably with N-bromosuccinimide. Amine, thiol and sulphite ligands were attached efficiently and in high densities (1–1.5 mmol/g dry) to the brominated matrices, using concentrated reaction mixtures. Organic solvents were not required. Bromohydroxypropyl matrices and amine or carboxylate derivatives could thus be used to produce matrices for all major forms of adsorption chromatography. This chemistry was used to prepare high-density mercaptoalkyl acid matrices, which were successfully applied to mixed mode purification of crude chymosin and compared with matrices used previously.