The accessibility of the prosthetic group biotin during monomeric avidin affinity chromatography.
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Avidin↗
Biomedical subjects
A W Purcell
Publications and source records attributed to A W Purcell.
Nonprecipitating anti-La(SS-B) autoantibodies in primary Sjögren's syndrome.
Anti-La(SS-B) precipitin-negative sera show a restricted epitope recognition and can be easily overlooked in routine laboratory testing. We have therefore determined the prevalence of nonprecipitating anti-La(SS-B) antibodies in patients with primary Sjögren's syndrome and studied their clinical and immunological associations. Clinical details were obtained from 68 patients with primary Sjögren's syndrome, and serum samples were examined by enzyme-linked immunosorbent assay using purified recombinant La, 60-kDa Ro, and 52-kDa Ro proteins and by counterimmunoelectrophoresis. Thirteen patients (19%) were identified with anti-La antibodies which were nonprecipitating. These patients had similar clinical findings to other groups of patients with Sjogren's syndrome, but had significantly lower rheumatoid factor and serum IgG levels than patients with anti-La precipitins. None of the patients with nonprecipitating anti-La antibodies had previously contained anti-La precipitins in their sera. Furthermore, they tended to have lower levels of antibodies directed against denatured 60-kDa Ro (but not 52-kDa Ro) compared with anti-La precipitin-positive patients. Patients with Sjögren's syndrome associated with nonprecipitating anti-La antibodies represent a stable serological and clinical subset in which there appears to be limited diversification of the autoimmune response to the Ro60 and La proteins of the Ro/La ribonucleoprotein.
Antibodies, Antinuclear↗
Conformational effects in reversed-phase high-performance liquid chromatography of polypeptides. II. The role of insulin A and B chains in the chromatographic behaviour of insulin.
The contribution of the insulin A- and B-chain to the retention and bandwidth behaviour of bovine insulin has been investigated. The influence of temperature and residence time on the logarithmic capacity factor (log k) versus the mole fraction of organic modifier psi, i.e. the effect of temperature and ligand residency on the S and log k0 values of the individual peptide chains, were assessed at temperatures between 5 and 85 degrees C and elution times between 30 to 90 min with an n-octadecyl (C18) and an n-butyl (C4) sorbent. Analysis of these log k versus psi dependencies revealed that the insulin A-chain exhibits retention behaviour significantly different to the intact insulin molecule whilst the B-chain exhibits retention behaviour which is remarkably similar to the parent protein. However, in terms of kinetic processes, the A-chain exhibited a peak-splitting phenomenon at higher temperatures which was similar to the behaviour of the intact insulin molecule, whilst only bandbroadening with no peak splitting was apparent for the B-chain. Overall, the similarity of the retention behaviour of the insulin B-chain and the intact insulin molecule with regard to their temperature and residency dependencies suggests that the insulin B-chain makes a significant contribution to the chromatographic contact region of the insulin molecule when this polypeptide is exposed to hydrocarbonaceous ligands at low to intermediate temperatures due to the progressive unfolding of the molecule and greater accessibility of the previously buried B-chain residues.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
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.
Amino Acid Sequence↗
Identification and synthesis of altered peptides modulating T cell recognition of a synthetic peptide antigen.
In studies of T cell responses to synthetic peptides we have observed agonist and antagonist activities associated with contaminants identified within the parent synthesis. The synthesis of two candidate analogues implied by a peptide contaminant formed during the synthesis of La 51-58 (IMIKFNRL) has been carried out. The peptide contaminant was 17-18 Da smaller than the parent peptide consistent with a modified asparagine residue at position 6 and so we synthesised both an aspartimide and a nitrile analogue, representing cyclisation or dehydration of the asparagine residue. The candidate aspartimide and nitrile analogues both bound empty MHC class I molecules to form allo determinants recognised by monoclonal antibodies. These results demonstrate that altered synthetic peptides can bind class I MHC molecules and prompt caution in the use of synthetic peptides as a source of immunising antigen.
Amino Acid Sequence↗
High-performance liquid chromatography of amino acids, peptides, and proteins. 123. Dynamics of peptides in reversed-phase high-performance liquid chromatography.
The dynamics of several peptides in reversed-phase high-performance liquid chromatography (RP-HPLC) have been investigated on both n-octadecyl (C18) silica and n-butyl (C4) silica sorbents. In particular, the conformational interconversions and the relative rates of chromatographic relaxation of bombesin, glucagon, and beta-endorphin on both C18 and C4 n-alkylsilicas were monitored by examining changes in the experimental bandwidths of these peptides as a function of temperature and column residence time under linear gradient elution RP-HPLC conditions. The observed band-broadening trends were correlated with previously derived retention parameters and thermodynamic descriptors of the association process determined for bombesin, beta-endorphin, glucagon, and a control peptide, penta-L-phenylalanine. This study confirms that bandwidth measurements can be used as an integral experimental component to study the effect of the secondary structure of peptidic solutes on their RP-HPLC retention behavior. Further, the data demonstrate the utility of RP-HPLC as a tool to examine peptide conformational dynamics at hydrophobic surfaces. The relevance of these results to the general phenomenon of peptide-lipid interactions is discussed in terms of the associated evidence for lipid-induced changes in the conformation of these three bioactive peptides.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
High-performance liquid chromatography of amino acids, peptides and proteins. XC. Investigations into the relationship between structure and reversed-phase high-performance liquid chromatography retention behaviour of peptides related to human growth hormone.
The gradient elution behaviour of eight synthetic peptides encompassing residues [6-13] of human growth hormone, i.e. Leu1-Ser-Arg-Leu-Phe-Asp-Asn-Ala8, has been investigated, by using an octadecylsilica, a butylsilica, and a polymeric fluorocarbon as stationary phases. Quantitative expressions, derived from the linear-solvent-strength theory and the general plate-height theory, were used to assess the influence of gradient time on the relative retention and bandwidths of these peptides. It was demonstrated that the chromatographic properties of the cyclised imide form involving Asp6 are consistent with the formation of a highly stabilised amphipathic helix, while the open-chain alpha- and beta-rearranged forms eluted as less rigid structures. The putative hydrophobic contact region consists of two leucine residues and one phenylalanine residue. From an analysis of the retention and bandwidth data obtained at pH 9, a surface-induced molecular reorientation of the beta-linked peptides was observed, in which the repulsion of the aspartyl carboxyl group from the hydrophobic stationary phase directs the C-terminal moiety away from the sorbent surface. Furthermore, the fluorocarbon sorbent exhibited characteristics favourable for use in preparative purification of these peptides. The present results demonstrate the sensitivity of reversed-phase high-performance liquid chromatography (RP-HPLC) to monitor small changes in the interactive behaviour of peptides with hydrocarbonaceous ligands and aquo-organic solvent combinations in reversed-phase systems. These observations further illustrate the general utility of HPLC for investigating the conformational behaviour of peptides at solid-liquid interfaces.
Amino Acids↗
High-performance liquid chromatography of amino acids, peptides and proteins. XCI. The influence of temperature on the chromatographic behaviour of peptides related to human growth hormone.
The influence of temperature on the gradient elution properties of synthetic peptides related to residues [6-13] of human growth hormone, e.g., Leu1-Ser-Arg-Leu-Phe-Asp-Asn-Ala8, has been studied by using both an octadecylsilica and a polymeric fluorocarbon stationary phase. Correlation of changes in the solute hydrophobic contact area and affinity for the stationary phase, as given by S and log k0 values respectively, revealed that the alpha- and imide forms are more conformationally stable than the beta-linked peptide. In addition, negative values of the standard entropy change, delta S0 assoc, for the transfer of the solute to the stationary phase, were observed for both alpha- and beta-linked peptides. These results are indicative of an increased ordering of the system upon solute adsorption and implies that the open-chain peptides exist in solution in more flexible conformations, while the helical structure of the cyclised imide is more rigid and constrained. The implications of the relative conformational stability of these peptides in their role as insulin-potentiating agents is also discussed.
Amino Acids↗
Induction of amphipathic helical peptide structures in RP-HPLC.
The retention behavior of a series of amphipathic peptide multimers based on the amino acid sequence [KSEEQLA]n has been investigated using reversed-phase high performance liquid chromatography (RP-HPLC). Structure-retention parameters which are related to the hydrophobic contact area and affinity of these peptides for the immobilized hydrocarbonaceous ligands were determined over a range of operating temperatures between 5 degrees and 85 degrees C. The influence of ligand hydrophobicity was assessed by comparison of peptide retention behavior using an n-octadecyl (C18)- and an n-butyl (C4)-silica of similar ligand density. The results demonstrated that ligand-mediated conformational effects can stabilize peptide structure depending on the chromatographic residence time and peptide length. In particular, more highly stabilized secondary structures were evident for the longer peptides. In addition, the amphipathic secondary structure of the peptides were more effectively stabilized by the more hydrophobic C18 ligands relative to the shorter C4 ligands. Additional information on the interactive dynamics of these peptide multimers was obtained from analysis of bandwidth dependencies under the different chromatographic conditions. These studies provide further insight into the role which hydrophobic forces can play in the stabilization of peptide structures.
Amino Acid Sequence↗