M.I. Aguilar

High-performance liquid chromatography of amino acids, peptides and proteins : LXVI. Investigations on the effects of chromatographic dwell in the reversed-phase high-performance liquid chromatographic separation of proteins

Abstract Polypeptide and protein retention on chemically modified hydrocarbonaceous surfaces such as n-alkylsilicas is generally assumed to be mediated by weak solvo-phobic effects with relatively rapid kinetics of adsorption and desorption. Recent studies have, however, indicated that multi-zoning effects can be generated for many proteins under certain chromatographic conditions. These effects can lead to asymmetric or multiple peaks for an apparently homogeneous polypeptide or protein. The present study examines retention models for the reversed-phase liquid chromatographic separation of polypeptides and proteins undergoing conformational changes in the mobile phase and at the stationary phase surface. In addition the influence of the chromatographic dwell or residence time on resolution and recovery is examined. Analytical expressions for the change in retention and peak shape have been used to evaluate the role of secondary dynamic effects which may lead to broad asymmetric peaks or multiple peaks for an apparently homogeneous protein chromatographed on n-alkylsilicas under defined chromatographic conditions. Methods of classifying protein retention behaviour in reversed-phase systems according to the characteristics of these multiphasic transitions are suggested.

High-performance liquid chromatography of amino acids, peptides and proteins CXV. Thermodynamic behaviour of peptides in reversed-phase chromatography☆

The thermodynamic behaviour of three peptides, bombesin, beta-endorphin and glucagon, was studied under reversed-phase high-performance liquid chromatographic conditions. Experimental data related to the interactive surface contact area (S values) and solute affinity (log k0) were derived over a range of temperatures between 5 and 85 degrees C. These experimental conditions allowed changes in the secondary structure of the solute to be monitored. The influence of the nature of the stationary phase ligand on the relative conformational stability of the three peptides was analysed by acquiring data with n-octadecyl silica (C18) and n-butyl silica (C4) sorbents. Values for the relative changes in entropy and enthalpy associated with the interactive process were also determined. The results provide further insight into the factors involved with the stabilization of secondary structure and the mechanism of the interaction of peptides with hydrophobic surfaces.

High-performance liquid chromatography of amino acids, peprtides and proteins : LXIX. Evaluation of retention and bandwidth relationships of myosin-related peptides separated by gradient elution reversed-phase high-performance liquid chromatography

Abstract The gradient elution behaviour of ten peptide analogues encompassing the primary amino acid sequences adjacent to the serine-19 phosphorylation site of myosin light chain has been investigated using a 30-nm pore diameter n-butilsilica stationary phase and 0.1% trifluoroacetic acid—water—acetonitrile mobile phases. Quantitative expressions derived from linear solvent strength theory and general plate height theory have been used to assess the influence of gradient time and flow-rate on the relative retentions and bandwidths of these peptides. The retention behaviour of these peptide analogues was found to closely mirror the predictions of linear solvent strength gradient elution theory. Although the experimentally observed bandwidth changes over a conventional range of gradient conditions (i.e.tG = 30–60 min) also correlated with bandwidth changes predicted on the basis of plate theory derived for low molecular weight organic molecules, for very steep and very shallow gradients divergencies from theory were observed. The significance of these discrepancies is discussed in relation to the influence of amino acid sequence changes rather than composition of these peptide analogues. These data allow the importance of sequential effects on the chromatographic behaviour and in particular, the resolution optimisation of these closely related peptides to be evaluated.

High-performance liquid chromatography of amino acids, peptides and proteins LXVIII. Evaluation of retention and bandwidth relationships of peptides related to luteinising hormone-releasing hormone and growth hormone-releasing factor, separated by gradient elution reversed-phase high-performance liquid chromatography

Abstract The gradient-elution behaviour of six peptides related to luteinising hormone-releasing hormone and three peptides related to growth hormone-releasing factor has been investigated using a 30-nm pore diameter n-butylsilica stationary phase and water—acetonitrile mobile phases containing 0.1 % trifluoroacetic acid. The effect of gradient time and flow-rate on the relative retentions and bandwidths of these peptides have been assessed using quantitative expressions, derived from linear solvent strength theory and general plate height theory. Whilst the retention data for these hormonal peptides appear to be adequately described by gradient elution theory for the range of operational conditions used in this study, the experimental band broadening data are not in agreement in all cases with bandwidths predicted on the basis of plate theory derived for low-molecular-weight organic molecules. Possible causes of the “non-ideal” peak shapes observed for these peptides are discussed.

High-performance liquid chromatography of amino acids, peptides and proteins CXXVIII. Effect of d-amino acid substitutions on the reversed-phase high-performance liquid chromatography retention behaviour of neuropeptide Y[18–36] analogues

The reversed-phase high-performance liquid chromatographic (RP-HPLC) gradient elution behaviour of a series of peptides related to Neuropeptide Y (NPY) has been investigated. The peptides studied included NPY, NPY[13-36], NPY[18-36] and a series of 16 analogues of NPY[18-36], each with a single D-amino acid substitution. Chromatographic parameters which relate to the interactive contact area and the binding affinity have been evaluated with two different stationary phase ligands and two organic modifiers. The results demonstrate that D-amino acid substitutions in the sequence region encompassing amino acid residues NPY[27-31] of these NPY[18-36] peptides significantly influence the interactive behaviour of these peptides relative to the unsubstituted NPY[18-36] molecule, while substitutions in the N- and C-terminal regions had little effect. Further, these results indicate that, in hydrophobic environments, NPY[18-36] adopts a significant degree of secondary structure which is severely disrupted by the presence of the D-amino acids in the central portion of the molecule.

Antimicrobial Peptide Structure and Mechanism of Action: A Focus on the Role of Membrane Structure

Antimicrobial peptides (AMPs) are showing increasing promise as potential candidate antibacterial drugs in the face of the rapidly emerging bacterial resistance to conventional antibiotics in recent years. The target of these peptides is the microbial membrane and there are numerous models to explain their mechanism of action ranging from pore formation to general membrane disruption. The interaction between the AMP and the target membrane is critical to the specificity and activity of these peptides. However, a precise understanding of the relationship between antimicrobial peptide structure and their cytolytic function in a range of organisms is still lacking. This is a result of the complex nature of the interactions of AMPs with the cell membrane, the mechanism of which can vary considerably between different classes of antimicrobia peptides. A wide range of biophysical techniques have been used to study the influence of a number of peptide and membrane properties on the cytolytic activity of these peptides in model membrane systems. Central to characterisation of this interaction is a quantitative analysis of the binding of peptide to the membrane and the coherent dynamic changes in membrane structure. Recently, dual polarization interferometry has been used to perform an in depth analysis of antimicrobial peptide induced membrane perturbation and with new mass-structure co-fitting kinetic analysis have allowed a real-time label free analysis of binding affinity and kinetics. We review these studies which describe multi-step mechanisms which are adopted by various AMPs in nature and may advance our approach to the development of a new generation of effective antimicrobial therapeutics.

High-performance liquid chromatography of amino acids, peptides and proteins. LXXXVI. The influence of different displacer salts on the retention and bandwidth properties of proteins separated by isocratic anion-exchange chromatography.

The influence of different displacer salts on the retention behaviour of seven globular proteins ranging in molecular weight from 12,000 to 69,000 was investigated using the Mono Q anion-exchange resin as the stationary phase. Isocratic retention data were collected using several different alkali metal halides as the displacing salt, thereby systematically varying the anion and cation species in the series F-, Cl- and Br- and Li+, Na+ and K+. The different anions were found to reduce protein retention in order of their decreasing hydrated ionic radii. Protein Zc values were found to be lower for fluoride and bromide than for chloride. It was demonstrated that the cationic co-ions also influence solute retention properties with this anion-exchange resin through, inter alia, preferential interactions with the protein solute. Protein band-broadening was found to systematically vary with the choice of displacer salt. These changes were related to known Hofmeister effects on protein aggregation kinetics and solubility and the degree of ion penetration at the double layer of the stationary phase-mobile phase interface. These studies now provide a rapid comparative basis for evaluating the mechanism of co- and counter-ion interactions with proteins in high-performance ion-exchange chromatographic systems.

High-performance liquid chromatography of amino acids, peptides and proteins CXXIX. Ceramic-based particles as chemically stable chromatographic supports☆

Porous zirconia based particles have been modified using different derivatisation procedures. The modified particles were characterised in terms of their accessible surface areas and degree of surface coverage of the bounded or physicoated phases utilising the strong and specific adsorption of phosphate ions to the zirconia surface. The hydroxyl group density was determined by a 1H NMR technique. The particles were modified by immobilising different silanes to introduce either hydrophobic ligands or reactive groups onto the zirconia surface. In the latter case, various ligands were then covalently attached to the activated supports. Using this type of modification, n-octadecyl- (C18), carbohydrate- and Cibacron Blue F3GA-modified zirconia particles were produced. Furthermore, polymeric coated particles were prepared either by using polybutadiene or by cross-linking the carbohydrate modified sorbents. The pH stability of the different sorbents were determined in batch experiments and under chromatographic conditions. The leakage of ligands was monitored by UV absorption and by employing radioactively labelled ligands. The performance of the C18 reversed-phase modified zirconia in packed columns was also used as an indicator of changes in the surface chemistry following pH stability tests. The experimental results indicate that the Cibacron Blue F3GA dye-modified sorbent was stable up to pH 10.5, the C18 reversed-phase packing up to pH 13 and the carbohydrate-bonded phase up to pH 12. These investigations substantiate the favourable chemical and physical characteristics anticipated for surface modified zirconias for potential use as chromatographic adsorbents.

Conformational effects in reversed-phase high-performance liquid chromatography of polypeptides. I. Resolution of insulin variants.

In order to further characterise the role of conformation in the retention behaviour of polypeptides and proteins in reversed-phase high-performance liquid chromatography (RP-HPLC), the chromatographic properties of four different insulins have been studied as a function of temperature (over the range 5-85 degrees C) and column residence time (over the range 10-60 min). The role of the ligand structure was also investigated by comparing results obtained with a n-octadecyl (C18) and a n-butyl (C4) ligand immobilised to the same porous silica. Comparative structure-retention-stability relationships were determined from an examination of the influence of temperature on a number of chromatographic parameters including the chromatographic contact area, the affinity constant and the experimental band width. The results demonstrated that variations in temperature can be used to affect significant changes in selectivity between the different insulins despite their very high degree of sequence homology. These observations have permitted specific amino acid residues, and in particular those residues encompassing the region A8-A10, to be proposed to be directly involved in the chromatographic contact area of the insulin molecules. Overall, the analysis of the changes in various chromatographic parameters in response to variation of the amino acid sequence, temperature and other experimental parameters provides a powerful tool to elucidate the structural basis for the interfacial stability and the role of conformation on the retention behaviour of polypeptides and proteins in RP-HPLC.

High-performance liquid chromatography of amino acids, peptides and proteins. LXXXIX. The influence of different displacer salts on the retention properties of proteins separated by gradient anion-exchange chromatography.

Abstract The influence of eight different displacer salts on the retention properties of four globular proteins, ranging in molecular weight from 14 000 to 43 000, was investigated by using the Mono-Q strong-anion-exchange resin as the stationary phase. Proteins were eluted under gradient conditions with a range of alkali metal halides to vary systematically the anion and cation species in the series F − , Cl − , and Br − and Li + , Na + and K + . Protein Z c values ( i.e. slopes of the ion-exchange retention plots, as derived from the dependency of the logarithmic capacity factor log k ′ on the concentration of the ionic displacer) generally increased when both the anion and cation were either chaotropic, e.g. KBr, or kosmotropic, e.g. NaF, in nature. Conversely, Z c values decreased when the displacer salt contained an anion-cation combination of a chaotropic and a kosmotropic ion, e.g. KF. These results indicate that the lyotropic properties of salts are additive in their effect on the interactive properties of proteins in anion-exchange chromatography. The Z c values were also found to depend on the manner in which the ionic strength was manipulated to affect elution, i.e. isocratic or gradient change in concentration of the displacing salt. Thus, isocratic experiments and gradient experiments with varied gradient time or varied flow-rate were observed to result in log k′ versus log 1/ c dependencies with non-coincident Z c values. The relationship between protein Z c values, the electrostatic contact area or ionotope, A c , and the electrostatic potential of the protein surface, ψ s , is discussed.