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M T Hearn

Publications and source records attributed to M T Hearn.

At least 73 records · Page 4Linked to original sources
PubMed1996

Influence of temperature on the retention behaviour of proteins in cation-exchange chromatography.

The chromatographic behaviour of several amino acid derivatives, peptides and proteins has been investigated with the so called "tentacle-type" LiChrospher-100 SO3- adsorbent and an analogous poly(2-sulphoethylaspartamide) cation-exchange adsorbent, PolySulphoethyl A. In particular, the dependences of the retention properties of a range of biosolutes on temperature and the chromatographic residence time were evaluated with the objective of gaining further insight into the influence of ligand type and flexibility and the role of solute conformation on the chromatographic behaviour of proteins with these two strong cation-exchange chromatographic adsorbents. The results indicate that significant differences in the chromatographic retention behavior between proteins and low-molecular-mass solutes occur as a function of temperature and the type of co- and counter ions present in the mobile phase with both adsorbents. Moreover, the dependences of the Zc and log Kc values on temperature for most of the proteins examined exhibited significant changes in magnitude between 4 and 75 degrees C, whilst no equivalent changes were evident for low-molecular-mass solutes. With the "tentacle-type" LiChrospher-1000 SO3- adsorbent at higher temperatures, e.g., at 75 degrees C, most of the proteins could still be eluted although several exhibited very large increases in their retention parameters. With the PolySulphoethyl A adsorbent, on the other hand, none of the proteins examined were eluted at 75 degrees C. The results moreover indicate that hydrophobic interactions play an increasingly important role in protein retention with both types of ion-exchange adsorbents at higher temperatures, but are more dominant with the PolySulphoethyl A ligand. In general, the Zc values for the proteins with the "tentacle-type" LiChrospher-1000 SO3- adsorbent were greater than those obtained with the PolySulphoethyl A adsorbent, suggesting that the "tentacular" ligands present on this strong cation-exchange adsorbent interact with protein molecules through larger contact areas. Collectively, these investigations provide further support for the concept that the adsorption behaviour of proteins with the "tentacle-type" LiChrospher-1000 SO3- adsorbent and similar types of "tentacular" ligand systems involves a multilayer dissolution mechanism with the protein interacting with a more diffuse or extended Donnan double layer in the ion-exchange environment, resulting in multi-site binding processes.

Absorption↗
PubMed1996

The disulphide bond structure of thyroid-stimulating hormone beta-subunit.

Previously only one of the six disulphide bonds within the beta-subunit of bovine thyrotropin (bTSH beta) has been unequivocally assigned. In the present investigation, the fluorescent alkylating reagent 5-N-[(iodoacetamidoethyl)amino]naphthalene-1-sulphonic acid has been employed as part of a double-alkylation strategy to allow the relative reactivities and the location of the six disulphide bonds of bTSH beta, after selective reduction, to be assigned by using reversed-phase HPLC peptide mapping techniques and associated methods of structural analysis. The most reactive disulphide bond was Cys88-Cys95; the second most reactive group of disulphide bonds involved the half-cystine residues Cys16, Cys19, Cys67 and Cys105 with the experimental results consistent with the assignment of disulphide bonds to Cys16-Cys67 and Cys19-Cys105. The least reactive group of half-cystine residues consisted of Cys2, Cys27, Cys31, Cys52, Cys83 and Cys85. The isolation, by high-performance ion-exchange chromatography, of a partly reduced bTSH beta derivative in which only the half-cystine residues Cys31, Cys85, Cys88 and Cys95 were labelled enabled the assignment of a previously uncharacterized disulphide bond to Cys31-Cys85. The remaining two assignments, Cys2-Cys52 and Cys27-Cys83, were made by comparison with the recently published human chorionic gonadotropin crystal structure. The flexibility of the double-labelling approach used in these studies demonstrates that only very small quantities are required for proteins containing an extensive number of half-cystine residues such as TSH beta, owing to the combination of the high resolution of the reversed-phase HPLC peptide mapping procedures and the sensitivity of the fluorimetric detection method.

Alkylation↗
PubMed1996

Application of a chromogenic bioassay procedure for the measurement of the proliferation of endothelial cells in vitro under the influence of the effects of steroid hormones and growth factors.

Development of in vitro procedures which permit the measurement of the synergistic biological influences of polypeptide growth factors and steroid hormones on cellular processes represents an important objective in reaching an understanding of the molecular basis of many physiological events, including those associated with the proliferation of endothelial cells and the subsequent angiogenesis which occurs, for example, during the estrous cycle and wound repair. The present investigations were undertaken to examine the use of nonradioisotopic procedures for the bioassay of polypeptide growth factors, such as basic fibroblast growth factor (bFGF), known to exert effects on the proliferation and migration of endothelial cells, and to overcome some of the recognised sensitivity limitations with endothelial cell proliferation assays based on the [3H]thymidine uptake methods due to the cell passage number and culture conditions. The experimental results confirm these objectives with establishment of a simplified chromogenic procedure for the sensitive determination of the effects of the steroid hormones, estradiol and progesterone, on the proliferation of endothelial cells in culture, either directly or via mediation of the effects of bFGF at low concentration levels. This procedure has provided experimental data suggesting that a hitherto unrecognised synergy may exist between these steroids and bFGF, with both estradiol and progesterone significantly antagonising the bFGF-stimulated proliferative response with bovine aortic endothelial cells (BAEC) in culture. Progesterone alone was also capable of directly stimulating BAEC proliferation, albeit transiently. Neither estradiol or progesterone affected the proliferation of the control 3T3 fibroblast cell population, in the presence or absence of a stimulatory dose of bFGF.

3T3 Cells↗
PubMed1996

Porous zirconia: a new support material for enzyme immobilization.

Four different proteases (trypsin, chymotrypsin, papain and pepsin) were covalently attached to the surface of a new type of porous zirconia, as well as a conventional porous silica, activated with 3-isothiocyanatopropyltriethoxy silane (NCS-silane). The immobilization efficiency onto the porous zirconia material was evaluated in terms of the amount of enzyme attached to the particles and from the biological activity remaining after the immobilization step. The results were compared with the corresponding experiments with a porous silica of similar surface area/g support material. In addition, the storage stability of the modified zirconia and silica biocatalysts were evaluated. These results indicated that specific immobilized enzyme biocatalysts can be achieved with this new zirconia support material which exhibits different properties to those observed with the more conventional silica-based materials. Moreover, the results with the enzyme-zirconia biocatalysts also indicate different characteristics when compared with data for the same enzymes immobilized under similar buffer conditions to organic support materials as previously described by various other investigators. The advantages of zirconia-based immobilized enzyme biocatalysts in terms of their density and chemical robustness are also described relative to other alternative support materials currently in use.

Chymotrypsin↗
PubMed1996

Application of immobilized metal ion chelate complexes as pseudocation exchange adsorbents for protein separation.

The interactions of horse muscle myoglobin (MYO), tuna heart cytochrome c (CYT), and hen egg white lysozyme (LYS) with three different immobilized metal ion affinity (IMAC) adsorbents involving the chelated complexes of the hard Lewis metal ions Al3+, Ca2+, Fe3+, and Yb3+ and the borderline Lewis metal ion Cu2+ have been investigated in the presence of low- and high-ionic strength buffers and at two different pH values. In contrast to the selectivity behavior noted with buffers of high ionic strength, with low-ionic strength buffers, these three proteins interact with the hard metal ion IMAC adsorbents in a manner more characteristic of cation exchange behavior, although in contrast to the cation exchange chromatography of these proteins, as the pH value of the elution buffer was increased, the retention also increased. The selectivity differences observed under these conditions appear to be due to the formation of hydrolytic complexes of these immobilized metal ion chelate systems involving a change in the coordination geometry of the im-M(n+)-chelate at higher pH values. The experimental observations have been evaluated in terms of the effective charge on the immobilized metal ion chelate complex and the charge characteristics of the specific proteins.

Adsorption↗
PubMed1996

Expression of fibroblast growth factor-8 in adult rat tissues and human prostate carcinoma cells.

Androgens are essential for normal prostatic and testicular function. However, paracrine and/or autocrine actions of a number of growth factors have been implicated in the function of these tissues. A recent addition to the fibroblast growth factor family, the so called androgen-induced growth factor (AIGF) or fibroblast growth factor-8 (FGF-8), has been proposed to be under strict androgen regulation and induction in the mouse mammary carcinoma cell line SC3. FGF-8, therefore, may have a local role in the prostate, which is known to be an androgen-responsive organ. This study reports, for the first time, the presence of FGF-8 mRNA in normal adult rat tissues (heart, brain, lung, kidney, testis, prostate and ovary), using an optimised reverse transcription and nested polymerase chain reaction (RT-PCR) procedure, although androgen-dependent FGF-8 expression was not demonstrated in these adult tissues. Consistent with the oncogenic characteristics of FGF-8, the corresponding mRNA was detected in the human prostate tumour cell lines LNCaP and DU145. Because the DU145 cell line is known to be androgen-independent, and the expression of FGF-8 mRNA in cultured LNCaP cells also occurred in the absence of exogenous androgens, it can be concluded that the expression of FGF-8 mRNA in these human cell lines, in the rat prostate and in other rat tissues is not under the regulation of androgens as hitherto proposed.

Androgens↗
PubMed1996

Conformational analysis of human growth hormone [6-13] peptide analogues.

The conformational analysis of a series of ten hGH[6-13] peptide analogues is reported. As part of our earlier studies, the alpha-aminosuccinimide modified fragment Asu11-hGH[6-13] has previously been identified as a potentiator of insulin activity in intravenous insulin tolerance tests, and various analogues have been subsequently designed, synthesised and employed to acquire structure-activity data. These studies have lead to the conclusion that the conformational characteristics at the C-terminus of each of the active peptide analogues is important to the biological activity. In the present investigation, molecular dynamics and simulated annealing techniques have been used to examine the accessible conformational states of the C-terminal region of ten different hGH[6-13] peptide analogues. Of these six are active peptide analogues while the other four show no biological activity. Examination of the conformer groups identified using this molecular dynamics approach showed a common conformational motif for each of the active peptides.

Growth Hormone↗
PubMed1996

Localization of basic fibroblast growth factor mRNA (FGF-2 mRNA) in the uterus of mated and unmated gilts.

In mated sows, the level of placental vascularization has a direct effect on fetal growth and litter birth weight. Vascularization of the endometrium and uterus under the control of various polypeptide growth factors is an important early stage in this process. Basic fibroblast growth factor (FGF-2), a polypeptide distributed throughout the mesodermal and neuroectodermal tissues of many species, is a vascular endothelial cell mitogen in vitro and has been implicated in neovascularization and wound healing in vivo. As part of our studies of the distribution of FGF-2 in uterine tissue and its role in placental development and embryo implantation, the localization and changes in the abundance of porcine FGF-2 mRNA in the uterus of mated and unmated gilts were investigated by in situ hybridization procedures. These procedures were based on the use of [alpha35S]-dATP-labeled oligonucleotide probes and a novel set of digoxigenin-labeled oligonucleotide probes generated by reverse transcriptase-polymerase chain reaction (RT-PCR) methods and anti-sense labeling strategies from the corresponding mRNA templates. With these in situ hybridization procedures, porcine FGF-2 mRNA was localized during the first 30 days of pregnancy to specific tissue areas in the porcine uterus comprising glandular and luminal epithelial cells and stromal cells of both the stratum functionalis and stratum basalis regions of the endometrium, and within the smooth muscle of myometrium and the associated blood vessels. However, no significant increase in the level of FGF-2 mRNA within these tissues was detected during these early stages of pregnancy or during the estrous cycle of unmated gilts. These distribution and abundance patterns are only partially compatible with other recent observations suggesting a possible role for changing levels of the mature polypeptide form of FGF-2 in the reproductive tract of sows during the early stages of pregnancy.

Animals↗
PubMed1996

Aberrant FGF-2, FGF-3, FGF-4 and C-erb-B2 gene copy number in human ovarian, breast and endometrial tumours.

The important role of oncogene amplification and tumour suppressor gene deletion in human tumours is becoming increasingly apparent. However, extensive screening of human tumours is required before the prognostic significance of such genetic abnormalities can be fully appreciated. The present investigation describes a rapid non-radioactive and largely automated procedure for the analysis of aberrant gene copy number in large numbers of tissue samples of different human tumours. This procedure is based on the sequential use of the polymerase chain reaction (PCR) and high performance ion exchange liquid chromatography (HPIEX). Using this rapid PCR/HPIEX technique, we have identified amplification and deletion of the FGF-2 gene and the FGF-3, FGF-4 and c-erb-B2 oncogenes in human tumours of the breast, ovary and endometrium. Comparison of the data with tumour pathology has revealed possible associations between aberrant gene copy number and tumour type, invasiveness and metastases.

Blotting, Southern↗
PubMed1995

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↗
PubMed1995

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↗
PubMed1995

Immunochemical characterization of two thyroid-stimulating hormone beta-subunit epitopes.

The epitopes of human thyroid-stimulating hormone (hTSH) recognized by two murine monoclonal antibodies (MAbs), designated MAb 279 and MAb 299, have been characterized. These MAbs are highly specific for the beta-subunit of TSH. The epitope recognized by MAb 279 appears to be completely conserved between bovine and human TSH and partially conserved in the porcine species. The TSH beta-subunit epitope recognized by MAb 299 is only partially conserved between the human, bovine and porcine species. Both MAbs are capable of inhibiting the binding of TSH to its receptor in a TSH radioreceptor assay, indicating that the epitopes either coincide or are located close to the TSH beta-subunit receptor-binding sites. The carbohydrate moieties of the TSH beta-subunit appear to play little or no role in the epitope recognition by MAb 279 or MAb 299 while the integrity of the disulphide bonds are essential. The epitopic recognition may also involve lysine residues, as determined by the immunoreactivity with both MAbs following citraconylation of TSH. In addition, the amino acid sequence region between residues bTSH beta 34-44 could be excised by trypsin digestion of bovine TSH beta (bTSH beta) without eliminating epitopic recognition by either MAb. These results provide further insight into the relationship between the structure of the TSH beta-subunit epitopes and location of the receptor-binding sites.

Alkylation↗
PubMed1995

Delineation of tyrosine-containing epitopes within the beta subunit of bovine thyrotropin.

The epitopes recognised by two monoclonal antibodies (mAb 279 and mAb 299), specific for the beta subunit of bovine thyroid-stimulating hormone (bTSH), have been localised using a technique in which the tyrosine residues in the bTSH beta subunit were subjected to modification when the bTSH beta subunit was complexed with either mAb or in the free, unbound state. The epitope recognised by mAb 279 was localised to the C-terminal region of bTSH beta with the tyrosine residue Tyr104 protected from modification by the presence of this mAb. In addition, the experimental results indicate that the tyrosine residues Tyr18 and/or Tyr112 are also involved in the mAb 279 epitope. The epitope recognised by mAb 299 was localised to the region 59-74 of bTSH beta as both Tyr59 and Tyr74 were protected from modification by the presence of this mAb. Since both mAbs have been previously found to inhibit receptor binding, the sequence regions/amino acid positions recognised by these mAbs are likely to represent determinants for receptor binding. Moreover, these data indicate that the identified amino acid residues are located on the surface of the molecule, consistent with predictions of the tertiary structure of the bTSH beta subunit based on the recently elucidated X-ray crystal structure of human chorionic gonadotropin.

Amino Acid Sequence↗
PubMed1995

High-performance liquid chromatography of amino acids, peptides and proteins. CXXXVIII. Adsorption of horse heart cytochrome c onto a tentacle-type cation exchanger.

Determination of the change in the Gibb's free energy from the adsorption isotherm associated with the interaction between a biomolecule and an ion-exchange resin is often achieved by assuming that a Langmuirean model prevails. However, the adsorption of horse heart cytochrome c onto the tentacle-type cation exchanger LiChrospher 1000 SO3- at pH 4.00 showed an isotherm of rectangular form. In this case the Langmuirean model is not applicable. In this paper, we propose an alternative way to deal with this situation, whereby the adsorption capacity of the adsorbent with a defined protein sample is studied as a function of displacing-ion concentration. The experimental conditions over defined ranges are then selected in order to relate this function to the change in the Gibb's free energy for the interaction between the protein and the ion exchanger. Additional comments about the general utility of the on-line adsorption vessel system employed to determine the adsorption isotherms are also made.

Adsorption↗
PubMed1995

High-performance liquid chromatography of amino acids, peptides and proteins. CXXXIX. Impact of operating parameters in large-scale chromatography of proteins.

Large-scale chromatography has been playing an important role in downstream treatment processing in biotechnology. In order to improve the productivity, the throughput of the chromatographic equipment was often increased by increasing the flow-rate and/or by increasing the column sample loading. This paper reports the results of a study on the impact of these and other operating parameters in affinity and ion-exchange chromatographic columns when used for protein purification. A sectional model was developed to predict protein adsorption processes in a packed column. The formulations of this mathematical model are presented in the Appendix. The present study was carried out with computer simulation based on this model and using data obtained from laboratory-scale columns. This model can simulate both the adsorption and washing stages of the protein purification process for both porous and non-porous particles. The effects of changing operating parameters were simulated and contour plots were generated for the easy identification of these effects. It was shown that both flow-rate and column loading can have a considerable impact on the processing rate and the yield of the column. As for the column capacity utilization, the impact of changing flow-rate is not significant at column loading of less than 80% in the test case. It was suggest that the present investigation provides a systematic predictive strategy which will greatly reduce the need for expensive, labour-intensive and time-consuming experimental work during process scale-up.

Adsorption↗
PubMed1995

Stability studies on maize leaf phosphoenolpyruvate carboxylase: the effect of salts.

The solution stability of phosphoenolpyruvate carboxylase (PEPC) has been determined in the presence of various salts by temperature-accelerated enzyme inactivation and also by using high-performance size-exclusion chromatography. Kosmotropic (water structuring) anions in the Hofmeister series (HPO(4)2-, citrate3-, SO(4)2-, F-, OAc-) and glutamate stabilized the enzyme most effectively, while Cl- (a borderline Hofmeister anion) and Br- (a chaotropic anion) were destabilizing. The effects of the cations on PEPC stability ranged from relatively inert (Na+, K+) to destabilizing ((CH3)4N+, NH4+, Li+). The observed stabilization of PEPC by specific salts has been interpreted in terms of the positive surface tension increment and the water-structuring effects conferred on the solution by the specific stabilizing reagents. Both these effects enhance hydrophobic interactions of proteins and increase the energy required to enlarge the surface area of the solvent cavity in which the protein resides. The destabilization of PEPC by some salts at a concentration of 0.5 M was associated with the dissociation of the tetrameric enzyme into its dimeric and monomeric forms, a process most probably occurring as a result of ion-peptide dipole binding, which promotes protein-solvent interaction and a subsequent reduction in the free energy of cavity formation. The stabilization of enzyme activity by kosmotropic salts depended on the salt concentration with maximum stabilization of PEPC in solution at 52 degrees C observed with 0.6-0.8 M sodium glutamate, 2 M KF, and 2.2 M KOAc. Higher concentrations of these salts resulted in decreased activity. This reduction in activity of PEPC in the presence of high concentrations of kosmotropic salts appears to be associated with irreversible conformational changes of the tetrameric enzyme.

Chromatography, High Pressure Liquid↗
PubMed1995

Protein selectivity in immobilized metal affinity chromatography based on the surface accessibility of aspartic and glutamic acid residues.

The interaction of different species variants of cytochrome c and myoglobin, as well as hen egg white lysozyme, with the hard Lewis metal ions Al3+, Ca2+, Fe3+, and Yb3+ and the borderline metal ion Cu2+, immobilized to iminodiacetic acid (IDA)-Sepharose CL-4B, has been investigated over the range pH 5.5-8.0. With appropriately chosen buffer and metal ion conditions, these proteins can be bound to the immobilized Mn+-IDA adsorbents via negatively charged amino acid residues accessible on the protein surface. For example, tuna heart cytochrome c, which lacks surface-accessible histidine residues, readily bound to the Fe3+-IDA adsorbent, while the other proteins also showed affinity toward immobilized Fe3+-IDA adsorbents when buffers containing 30 mM of imidazole were used. These studies document that protein selectivity can be achieved with hard-metal-ion immobilized metal ion affinity chromatography (IMAC) systems through the interaction of surface-exposed aspartic and glutamic acid residues on the protein with the immobilized Mn+-IDA complex. These investigations have also documented that the so-called soft or borderline immobilized metal ions such as the Cu2+-IDA adsorbent can also interact with surface-accessible aspartic and glutamic acid residues in a protein-dependent manner. A relationship is evident between the number of clustering of the surface-accessible aspartic and glutamic residues and protein selectivity with these IMAC systems. The use of elution buffers which contain organic compound modifiers which replicate the carboxyl group moieties of these amino acids on the surface of proteins is also described.

Animals↗