Biomedical subjects

M T Hearn

Publications and source records attributed to M T Hearn.

At least 145 records · Page 8Linked to original sources

PubMed1989

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↗

PubMed1989

Retention behaviour of paracelsin peptides on reversed-phase silicas with varying n-alkyl chain length and ligand density.

As part of further investigations on the characterization of the ligand-induced conformational stabilization of peptides, two series of n-alkyldimethylsilyl bonded silicas have been prepared. In series A the n-alkyl chain length, n, of the bonded phase was varied between 1 and 20 carbon atoms at a constant ligand density. In series B the ligand density, alpha exp, was gradually changed from 0 to 4.1 mumol/m2 on a C1, C4, C6, C8 and C18 bonded phase. The retention behaviour of four peptides of the paracelsin family were examined under isocratic conditions, using a ternary mobile phase of water-methanol-acetonitrile (22:39:39, v/v/v). Plots of k' versus n showed pronounced maxima between n = 2 and 4 carbon atoms, followed by a decrease by a factor of 3 at n = 5, whereas above 5 carbon atoms only a slight increase in k' was observed. The selectivity behaviour of the paracelsins A-D can be mainly rationalized by interaction of the amphiphathic polypeptide 3.6(13) (alpha)-helix with the hydrophobic ligand and protrusion of the key amino acid residues at positions 6 and 9 in the sequence. However, from experiments with a polystyrene stationary phase it is evident that hydrophobic interactions and different partition coefficients also contribute to the resolution of the paracelsin peptides. Furthermore, Van 't Hoff plots confirm significant free energy changes associated with retention. These observations provide the basis for evaluating the enthalpic and entropic changes associated with peptide interactions with n-alkyl silicas.

Amino Acid Sequence↗

PubMed1989

High-performance liquid chromatography of amino acids, peptides and proteins. XCII Thermodynamic and kinetic investigations on rigid and soft affinity gels with varying particle and pore sizes.

In these investigations Cibacron Blue F3GA was immobilized on soft gels, porous silicas, and non-porous glass beads. Hen egg white lysozyme, human serum albumin and yeast alcohol dehydrogenase were used as adsorbates with the dye-affinity sorbents. Batch experiments with continuous monitoring of protein concentration were employed to evaluate thermodynamic and kinetic behaviour of these proteins in finite bath systems. The observed adsorption kinetic rates of interaction of the above proteins with each of the dye-affinity sorbents were found to decrease with increasing protein molecular weight. Equilibration times, in the batch experimental mode, of the adsorption of lysozyme on the dye-affinity sorbents varied from 20 s for the non-porous glass beads with a size range of 20-40 microns to more than 60 min in the case of a porous sorbent with a particle diameter of 100-300 microns and 60 nm pore size. Furthermore, equilibration times, which represent the overall adsorption rates incorporating all the non-equilibrium effects, increased with all affinity systems when adsorption took place in the non-linear portion of the isotherm. The most dramatic increase was observed when sorbents with relatively high protein size to pore size ratios, lambda, were employed.

Alcohol Dehydrogenase↗

PubMed1989

High-performance liquid chromatography of amino acids, peptides and proteins. XCIII. Comparison of methods for the purification of mouse monoclonal immunoglobulin M autoantibodies.

A comparison of methods for the purification of naturally occurring mouse monoclonal autoantibodies, of the immunoglobulin M (IgM) isotype, has been performed to determine the optimal strategies for the isolation of IgM from ascites fluid and in vitro tissue culture hybridoma supernatants. In order to quantify each purification procedure, the concentration of IgM in eluted fractions was determined by using a double-sandwich mu-chain-specific anti-IgM enzyme-linked immunosorbent assay, and the purity of the IgM was determined by a bicinchoninic acid-based protein assay and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The most efficient single-step purification was based on size-exclusion chromatography on high-resolution Superose 6 HR 10/30 fast protein liquid chromatography (FPLC) columns. This procedure resulted in recoveries of monoclonal IgMs of ca. 71-86% with purities between 68 and 86%. Single-step chromatography of monoclonal IgM, on Superose 6 FPLC columns resulted in a 21-fold purification of IgM, prepared by the in vitro culture of hybridoma cells in dialysis membrane. Size-exclusion chromatography, performed with Sephacryl S-300 columns, resulted in reduced resolution of monoclonal IgM, with yields of ca. 57-80% and purity of ca. 42-58% compared with the high-resolution Superose 6 FPLC columns. "Non-ideal" size-exclusion chromatography on Superose 6 FPLC columns resulted in selective retention of monoclonal IgMs and elution of IgM with high-ionic-strength buffers in the trailing peak. Recovery of IgM with this strategy was high (ca. 82-92%) but the purity was not comparable to the single-step fractionation of IgM on Superose 6 FPLC columns. Single-step anion- and cation-exchange and mixed-mode hydroxyapatite chromatography resulted in only partial purification of monoclonal IgM with the applied procedures. With these latter separation techniques, monoclonal IgM was eluted with a variety of other ascites fluid or supernatant proteins, including those with apparent molecular weights identical to those of mouse IgG and albumin. Sequential purification of monoclonal IgMs by Mono Q anion exchange, followed by Superose 6 FPLC columns, resulted in a 2- to 3-fold purification of IgM but did not separate IgM from high-molecular-weight contaminants with apparent molecular weights similar to those of alpha 2-macroglobulin and IgG. Enrichment of monoclonal IgM from ascites fluid by ammonium sulphate precipitation revealed increasing IgM recovery with increasing ammonium sulphate final concentrations up to 60%.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗

PubMed1989

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.

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 Zc values (i.e. slopes of the ion-exchange retention plots, 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, Zc 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 Zc 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 l/c dependencies with non-coincident Zc values. The relationship between protein Zc values, the electrostatic contact area or ionotope, Ac, and the electrostatic potential of the protein surface psis, is discussed.

Amino Acids↗

PubMed1989

High-performance liquid chromatography of amino acids, peptides, proteins and polynucleotides. XCIV. Solid-phase hybridization of complementary oligonucleotides.

The ability of synthetic oligonucleotides to specifically hybridize to a complementary oligonucleotide immobilized on an anionic stationary phase has been investigated. A sigmoidal melting curve was obtained when oligonucleotide duplex formation on the column was plotted against hybridization stringency over the ionic strength range 0.2-0.42 M. These studies confirm a rapid method for determining the relative melting temperature of hybridized oligonucleotide complexes and provide a basis for the selection of stringency conditions optimal for various synthetic oligonucleotide probes.

Amino Acids↗

PubMed1989

Rapid chromatographic isolation and immunoblot characterization of immunoreactive fibroblast growth factor-related polypeptides from various tissues.

Procedures to rapidly isolate fibroblast growth factor (FGF)-like activity from a number of tissue sources (lung, plasma, brain, ovary, corpus luteum, pituitary, chondrosarcoma) of bovine, porcine or rat origin are described. In addition, immunoblotting experiments using well characterized and specific rabbit polyclonal anti-fibroblast growth factor beta (anti-FGF-beta) sera have been performed. Besides documenting the first report of the isolation of FGF-beta from bovine lung and plasma, these studies provide evidence for the existence of higher-molecular-mass proteins with FGF-beta-like immunoreactivity. For example, in addition to new truncated forms of the acidic and basic FGF (FGF-alpha and FGF-beta), respectively, other higher-molecular-mass immunoreactive proteins were detected in bovine, pig and rat brain, and in rat chondrosarcoma. The tissue distribution of these immunoreactive proteins and their competitive inhibition characteristics mitigate against the possibility that the polyclonal antisera are cross-reacting non-specifically with common cellular proteins. Rather, the data suggest that the immunoblotting technique is either detecting other proteins structurally related to FGF-beta or alternatively FGF-beta is strongly bound to specific carrier proteins (e.g. heparan sulphate proteoglycan fragments) associated with their transport and recognition at the cellular level.

Animals↗

PubMed1989

High-performance liquid affinity chromatography with phenylboronic acid, benzamidine, tri-L-alanine, and concanavalin A immobilized on 3-isothiocyanatopropyltriethoxysilane-activated nonporous monodisperse silicas.

Nonporous, microparticulate, monodisperse silicas with particle diameters between 0.7 and 2.1 microns are introduced as stationary phases in high-performance affinity chromatography. The immobilization of m-aminophenylboronic acid, p-aminobenzamidine, tri-L-alanine, and concanavalin A onto these silicas was successfully achieved using 3-isothiocyanatopropyl-triethoxysilane as an activation reagent. Immobilized phenylboronic acid was applied to the isolation of nucleosides, nucleotides, and glycoprotein hormones such as bovine follicotropin and human chorionic gonadotropin, while immobilized benzamidine was employed for the isolation of the serine proteases thrombin and trypsin, immobilized tri-L-alanine for the separation of pig pancreatic elastase and human leukocyte elastase, and immobilized concanavalin A for the isolation of horseradish peroxidase. In all affinity chromatographic systems studied, the nonporous monodisperse silicas showed improved chromatographic performance compared to results obtained with porous silica supports using identical activation and immobilization procedures. Furthermore, frontal analysis was used as a method to evaluate the influence of experimental parameters on biological activity and accessible ligand densities. Only minor changes in bioactivity were found with the nonporous affinity supports, where accessibilities were typically higher than ca. 60%. The immobilization of affinity ligands onto porous supports as used in this and associated papers thus represents a successful general procedure for the preparation of stable matrices with fast kinetics for use in high-performance affinity chromatography.

Alanine↗

PubMed1989

Gamma globulin-derived standards for the determination of molecular weights, transfer, and immunodetection efficiencies in protein blotting procedures.

Molecular weight markers which are detectable using labeled antispecies antibodies or labeled Protein A have been prepared for use as standards on protein blots. The standards were prepared by the controlled reduction followed by subsequent alkylation of gamma globulin. Separate sets of standards were prepared using gamma globulins derived from human, mouse, rabbit, and sheep species. Standards were also prepared using monoclonal-derived gamma globulins from human myeloma fluid and mouse ascites fluid. Standards produced from monoclonal-derived gamma globulins produced very sharp bands on sodium dodecyl sulfate-polyacrylamide gels and proved to be excellent standards for this technique alone. However, the markers were uniquely suitable for use as standards in protein blotting procedures because their detection was achieved by the procedure used to detect the transferred antigen(s). The detection of immunoglobulin G (IgG)-derived standards on protein blots from all the species listed above was demonstrated using appropriate horseradish peroxidase (HRP)-conjugated antispecies antibodies. The use of other detection systems (biotin-labeled antibody and subsequent detection with HRP-steptavidin, HRP-Protein A) was also validated with human IgG-derived standards. Furthermore, the standards were shown to be suitable for use on both nitrocellulose and cationized nylon-based supports and could be used when adjacent samples were run under reducing conditions. Hence the gamma globulin-derived standards serve as both a control to check the adequacy of transfer and immunodetection systems and as markers which enable the molecular weights of detected antigens to be calculated.

Animals↗

PubMed1989

Phosphorylation of superior cervical ganglion proteins during regeneration.

The incorporation of radioactive phosphate into proteins of both normal and regenerating ganglia of the sympathetic nervous system of the rat is reported. The incorporation reactions were carried out in vitro by incubating homogenates of excised ganglia with [gamma-32P]ATP under various conditions. It was found that incorporation of phosphate into proteins of regenerating ganglia in the molecular mass range 10,000-100,000 daltons increased up to 40% over incorporation into proteins from control ganglia during the first 3 days following injury and returned to control levels after 14 days. Analysis of the proteins by two-dimensional electrophoresis revealed that only few, i.e., less than 20, became radioactively labelled in homogenates of superior cervical ganglia in the presence of Ca2+, and even fewer in the presence of cyclic AMP. Furthermore, all these proteins fell within a narrow pI range of 4-6. The growth-associated protein, variously designated GAP-43, B-50, F-1, and pp46, has an enhanced level of expression and phosphorylation in regenerating ganglia compared with controls at day 3. Injury also caused consistently higher levels of incorporation into two other proteins with molecular masses at positions 55,000 and 85,000 and pI values of 5.1 and 4.5, respectively; the former protein most probably is beta-tubulin. The fact that both proteins are found in the 15,000 g pellet after the tissue has been solubilized in 0.5% nonionic detergent indicates that they may indeed by components of filament assemblies. Thus, the results suggest that protein phosphorylation is a mechanism involved in cytoskeletal function in regenerating nerve.

Adenosine Triphosphate↗

PubMed1989

Isolation of inhibin alpha-subunit precursor proteins from bovine follicular fluid.

Two proteins with structural characteristics similar to peptide sequences identified in the inhibin alpha-subunit precursor sequence have been isolated from bovine follicular fluid. A side-fraction from the purification of bovine follicular fluid inhibin with high levels of inhibin immunoactivity relative to its inhibin bioactivity was fractionated through a sequence of procedures which included triazine dye affinity and phenyl-Sepharose chromatography, gel permeation chromatography on Sephadex G-100, reverse phase HPLC, and preparative polyacrylamide gel electrophoresis. The first of the two proteins identified had a molecular mass of 25-26K under reducing and nonreducing conditions and a NH2-terminal sequence identical to that of 43K inhibin alpha-subunit and showed minimal activity (less than 2% activity) compared with bovine 31K inhibin in either the inhibin in vitro bioassay or the RIA. These data suggest that this protein is the alpha 1-166 sequence of the bovine inhibin alpha-subunit (designated alpha N-subunit), most likely released after processing of either the inhibin alpha-subunit precursor or the 43K alpha-subunit involved in the conversion of 58K to 31K inhibin. The other protein identified (designated pro-alpha C-subunit) has a molecular mass of 27K under nonreducing conditions and 20K and 6K under reducing conditions. It is inactive in the in vitro bioassay, although highly reactive in the inhibin RIA, and has NH2-termini identical to the pro sequence of the inhibin alpha-subunit precursor and the 20K alpha-subunit sequence. These results suggest that pro-alpha C is a disulfide-linked structure and may represent an intermediate in the dimerisation of alpha- and beta-subunits to form inhibin while the alpha N-subunit is probably a proteolytic product of either the alpha-subunit precursor or 58K inhibin.

Animals↗

PubMed1988

High-performance liquid chromatography of amino acids, peptides and proteins. LXXXVII. Comparison of retention and bandwidth properties of proteins eluted by gradient and isocratic anion-exchange chromatography.

The high-performance ion-exchange gradient-elution behaviour of a range of globular proteins has been investigated, using a strong anion exchanger as the stationary phase and sodium chloride as the displacer salt. Deviations were observed between the Zc values obtained from isocratic experiments and from gradient experiments with varied gradient time and varied flow-rate. These results indicate that theoretical treatments which relate gradient and isocratic elution processes do not adequately describe the retention behaviour of protein solutes separated by ion-exchange methods. Furthermore, the experimentally observed bandwidths deviated significantly from values predicted on the basis of plate theory for low-molecular-weight molecules. The significance of these results is discussed in terms of the influence of experimental parameters on the ability of particular electrostatically interactive areas on the surface of protein solutes to control the thermodynamic and kinetic properties of these polyelectrolyte molecules during ion-exchange chromatographic processes.

Amino Acids↗

PubMed1988

High-performance liquid chromatography of amino acids, peptides and proteins. LXXXVIII. Calculation of the average distance between protein solutes and the stationary phase during isocratic anion-exchange chromatography.

This investigation deals with protein retention behaviour in high-performance anion-exchange chromatography in terms of the average distance of approach between the protein solute and the positively charged anion-exchange stationary-phase surface. The theoretical treatment is based on a modified Debye-Hückel theory for spherical impenetrable ions, where the electrostatic potential energy has been related to the chromatographic capacity factor, k'. Results are presented for three globular proteins, eluted isocratically from a Mono-Q strong anion-exchange resin with sodium chloride as the displacer salt by a mobile phase with pH in the range 5.50-9.60. Analysis of experimental retention data indicates that topographically predefined, charged regions on the protein surface, called ionotopes, control the orientation and approach distance of the protein solute.

Amino Acids↗

PubMed1988

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.

Amino Acids↗

PubMed1988

High-performance liquid chromatography of amino acids, peptides and proteins. LXXXV. Evaluation of the use of hydrophobicity coefficients for the prediction of peptide elution profiles.

The gradient elution behaviour of five synthetic decapeptide analogues has been investigated using an octadecylsilica stationary phase and trifluoroacetic acid-water-acetonitrile mobile phases. The influence of gradient time and flow-rate on the relative retentions and bandwidths of these peptides was assessed using quantitative expressions derived from linear solvent strength theory and general plate height theory. Linear relationships between logarithmic median capacity factors, log k, and the mole fraction of organic solvent modifier, phi, were observed over the experimental range of conditions used. The slopes of these plots were different for all peptides, which indicates that divergences will occur in the prediction of peptide retention times due to conformation dependent changes in hydrophobic contact area occupancy at the stationary phase surface. However, the differences in S values (tangent to the curve obtained in a plot of log k versus phi) for these peptides were not substantial enough to seriously affect the prediction of peptide retention times at one gradient slope from those observed at another. In addition, significant differences existed between experimental and theoretical peak capacity data of these peptide analogues of similar molecular weight and overall polarity, particularly at lower flow-rates or longer residence times. These results once again demonstrate that additional diffusional and interactive processes occur during the reversed-phase separation of peptides and proteins which are not yet adequately formalized by current chromatographic theory.

Amino Acids↗

PubMed1988

High-performance liquid chromatography of amino acids, peptides and proteins. LXXXIV. Application of derivative spectroscopy to the study of column residency effects in the reversed-phase and size-exclusion liquid chromatographic separation of proteins.

The influence of column residency times on the reversed-phase gradient elution behaviour of human and bovine growth hormones has been investigated using on-line photodiode array spectroscopic detection. Stationary phase induced effects on protein conformation were monitored by changes in the maxima to minima ratio of the second derivative spectrum of the eluted protein. Significant changes in the second derivative ratio of the unmodified and the fully reduced and alkylated protein were observed following long incubation times, i.e. t(dwell) greater than 15 min, at the stationary phase surface in the presence of 0.1% trifluoroacetic acid before elution with a 0-75% aqueous acetonitrile gradient. The application of multi-wavelength detection in the study of equilibrium unfolding of growth hormones in urea by size-exclusion chromatography was also investigated. On-line photodiode array instrumentation and derivative spectra rationing was employed to monitor tertiary and quaternary structural changes associated with protein denaturation during a chromatographic separation. These studies clearly demonstrate the powerful detection capabilities of such instrumental approaches for the on-line evaluation of both stationary phase surface and/or mobile phase mediated changes in protein conformation.

Alkylation↗

PubMed1988

Psychological correlates of differential infertility diagnosis in an in vitro fertilization program.

We describe a study designed to assess the role of infertility diagnosis in differentiating participants in an in vitro fertilization program on psychological variables. Three hundred forty-eight in vitro fertilization candidates representative of five diagnostic categories (tubal problems, endometriosis, male factor, multiple factors, and idiopathic) underwent psychological assessment before entering the in vitro fertilization program. Measures of personality functioning, depression, anxiety, social support, coping ability, and marital adjustment were obtained. Analyses revealed significant differences among diagnostic groups on several psychological measures. As well, candidates were categorized as having either organic or functional (idiopathic) infertility, and analyses revealed several differences between these two groups. Results suggest ways of providing individualized psychological support in vitro fertilization participants.

Adaptation, Psychological↗

PubMed1987

The isolation of polypeptides with FSH suppressing activity from bovine follicular fluid which are structurally different to inhibin.

Three proteins (31, 35 and 39 kDa) with inhibin-like activity have been isolated from bovine follicular fluid with identical NH2-terminal amino acid sequences. These polypeptides are distinct from inhibin, based on their different NH2-amino acid sequence, molecular masses, absence of a subunit structure, absence of inhibin immunoactivity and the failure of inhibin antiserum to neutralize their bioactivity in vitro. Their inhibin-like biological activities based on their ability to suppress FSH cell content by pituitary cells in culture are 5-10% of bovine 31 kDa inhibin.

Amino Acid Sequence↗