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Biomedical subjects

T A Bewley

Publications and source records attributed to T A Bewley.

At least 19 recordsLinked to original sources
PubMed1995

Surface denaturation at solid-void interface--a possible pathway by which opalescent particulates form during the storage of lyophilized tissue-type plasminogen activator at high temperatures.

During protein lyophilization, it is common practice to complete the freezing step as fast as possible in order to avoid protein denaturation, as well as to obtain a final product of uniform quality. We report a contradictory observation made during lyophilization of recombinant tissue-type plasminogen activator (t-PA) formulated in arginine. Fast cooling during lyophilization resulted in a lyophilized product that yielded more opalescent particulates upon long term storage at 50 degrees C, under a 150 mTorr nitrogen seal gas environment. Fast cooling also resulted in a lyophilized cake with a large internal surface area. Studies on lyophilized products containing 1% (w/w) residual moisture and varying cake surface areas (0.22-1.78 m2/gm) revealed that all lyophilized cakes were in an amorphous state with similar glass transition temperatures (103-105 degrees C). However, during storage the rate of opalescent particulate formation in the lyophilized product (as determined by UV optical density measurement in the 360 to 340 nm range for the reconstituted solution) was proportional to the cake surface area. We suggest that this is a surface-related phenomenon in which the protein at the solid-void interface of the lyophilized cake denatures during storage at elevated temperatures. Irreversible denaturation at the ice-liquid interface during freezing in lyophilization is unlikely to occur, since repeated freezing/thawing did not show any adverse effect on the protein. Infrared spectroscopic analysis could not determine whether protein, upon lyophilization, at the solid-void interface would still be in a native form.

Calorimetry, Differential Scanning↗
PubMed1991

Effect of freezing on aggregation of human growth hormone.

The effect of freezing on formation of soluble and insoluble aggregates of human growth hormone (hGH) was studied. The amount of soluble aggregates was affected very little by freezing regardless of the cooling rate. In contrast, the formation of insoluble aggregates (particulates), as determined by light scattering in the 340- to 360-nm range, was found to increase sharply with increasing cooling rates. The amount of these particulates was also dependent on the pH of the solution. Freezing hGH solutions formulated at pH 7.4 resulted in highly scattering solutions, whereas pH 7.8 formulations showed significantly less scattering. These results emphasize the importance of understanding the freezing phenomenon for protein solutions and suggest that the formation of soluble aggregates and insoluble particulates may have different mechanisms.

Freezing↗
PubMed1987

Elephant growth hormone. Isolation and characterization.

Growth hormone has been purified to homogeneity from elephant pituitary glands. It has 191 amino acids with two disulfide bridges and a single tryptophan residue. The somatotropin activity is only 15% when compared with the bovine hormone in the radioreceptor binding assay. From circular dichroism spectra alpha-helical content of elephant growth hormone is estimated to be 50%. Difference absorption spectra of the hormone suggest the presence of a hydrogen bond between the single Trp and a carboxylate ion.

Amino Acids↗
PubMed1987

Elephant prolactin: isolation and characterization.

Prolactin was isolated from anterior lobes of elephant pituitary glands. It consisted of 199 amino acids with three disulfide bridges and two tryptophan residues as found in prolactin from other species. The sequence of the NH2-terminal 28 amino acids was determined and shown homologous with the ovine hormone. In comparison with ovine prolactin, a marked difference was seen in the methionine content; the elephant hormone possessed only 18-34% lactogenic potency. The conformation of elephant prolactin was examined by zero order, second order and circular dichroism spectroscopy. The alpha helical content was estimated to be about 60%. In comparison with prolactins from other species, the second order spectra of elephant prolactin suggest that the local microenvironment for one or both tryptophan residues is somewhat different.

Amino Acids↗
PubMed1987

Conformation of sturgeon pituitary somatotropin.

Pituitary somatotropin (growth hormone) from the sturgeon (Acipenser gulden-stadti) has been studied by zero-order and second-order absorption spectroscopy, as well as by circular dichroism. Difference absorption spectra have also been generated during proteolytic digestion of the hormone. The molar extinction coefficient of the native protein was found to be 15,000 +/- 110 M-1 cm-1 at 278.5 nm. Comparison of the conformations of sturgeon somatotropin and somatotropins isolated from several mammalian species, including bovine and human, indicates a close relationship between these molecules. Such similarities may be related to the relatively high biopotency of this fish hormone in mammalian assay systems.

Animals↗
PubMed1987

Conformation of equine pituitary somatotropin.

Equine pituitary somatotropin (growth hormone) has been studied by zero-order and second-order absorption spectroscopy, and by circular dichroism. Difference absorption spectra have also been generated during proteolytic digestion of the hormone. The molar extinction coefficient of the native protein was found to be 16,050 +/- 330 M-1 cm-1 at 278.1 nm. Comparison of the conformations of equine somatotropin and somatotropins isolated from several other mammalian species indicates a close structural relationship between these molecules. With the increasing number of species which have been studied, it is becoming evident that with regard to conformation, the somatotropins can be subdivided into at least three major groups.

Animals↗
PubMed1986

The conformation of monkey pituitary somatotropin.

Monkey pituitary somatotropin has been studied by zero-order, second-order, and circular dichroism spectroscopy. Difference absorption spectra have also been generated during proteolytic digestion of the hormone. The molar extinction coefficient of the native protein was found to be 23,800 +/- 550 (M-1 cm-1) at 276.6 nm. A comparison of the conformations of monkey and human pituitary somatotropins indicates a close relationship between the two molecules, including alpha-helix contents of 55 +/- 5%.

Animals↗
PubMed1986

Structure-function studies on human growth hormone. Evidence that tertiary structure is essential for biological activity.

The relationship between the conformation of human pituitary growth hormone (hGH), biological activity, and ligand binding activity was studied by comparing conformational details previously published on in vivo and in vitro studies of identical samples of hGH and its known derivatives. In vivo assays included the rat tibia test for somatotropic activity and the pigeon crop-sac assay for lactogenic hormone activity. Relative binding affinities were compared in radioimmunoassays using 125I-hGH as tracer with 1) anti-human chorionic somatomammotropin (hCS) serum (low discriminatory hybrid assay), 2) anti-hGH sera (in conventional assays), 3) monospecific anti-hGH serum (absence of cross-reaction with hCS) and 4) human anti-hGH sera obtained from GH-deficient patients on replacement therapy. In addition, binding affinities were examined in two receptor-binding assays, one specific for somatotropic activity (rabbit liver membranes, 125I-hGH), and the other, for lactogenic hormones (rabbit mammary membranes, 125I-oPRL). The conformational properties of native hGH and various chemically and enzymatically modified derivatives of the hormone were evaluated primarily from circular dichroism spectra, while conformational stabilities were estimated from the relative rates of tryptic digestion. Unfragmented, but chemically modified derivatives, exhibited good parallelism between retention or loss of native conformation and the in vivo potencies and in vitro binding affinities. None of the fragments of hGH showed activity in any of the radioreceptor assays or radioimmunoassays. Two derivatives of hGH, which contain gaps of 6 or 12 residues in the polypeptide backbone produced by partial enzymatic digestion, had full or increased in vivo potencies, full activities in the radioimmunoassays, and were the most active derivatives in both radioreceptor assays. One of these, missing the hexapeptide corresponding to residues 135-140, was also found to retain nearly all the conformational properties of native hGH. These studies proved further evidence that 1) retention by modified forms of hGH of a high degree of in vivo biological potency or in vitro binding affinity is causally related to the retention of most of the conformation and conformational stability of the molecule, and 2) the biologically active, receptor-binding and immunoreactive sites on the hGH molecule are 3-dimensional in nature.

Animals↗
PubMed1985

Tertiary structure in deletion analogues of human beta-endorphin: resistance to leucine aminopeptidase action.

The presence of tertiary structure in aqueous solutions of the amino-terminal nine- and seventeen-residue analogues of human beta-endorphin has been further demonstrated by leucine aminopeptidase (LAP) cleavage of the amino-terminal tyrosine. The reactions were followed by difference absorption spectroscopy. While the amino-terminal pentapeptide showed no resistance to LAP action, the nonapeptide displayed definite resistance. The heptadecapeptide analogue was found to be completely refractory to LAP action under the conditions employed. Thermolysin cleavage of the Phe4-Met5 bond in the longer analogues destroys the tertiary structure, not only removing the previously reported red shift in tyrosine absorption but also resulting in complete normalization to LAP digestion.

Endorphins↗
PubMed1985

Conformation, and dissociation-recombination of chemically deglycosylated ovine lutropin.

The conformations of intact and acid dissociated, as well as the relative rates of conformational repair of reassociating, ovine lutropin, and a chemically deglycosylated form of lutropin, have been studied by circular dichroism, zero-order, and difference absorption spectroscopy. Deglycosylation produces only small conformational changes in the intact or acid dissociated form of the molecule. While conformational repair of reassociating lutropin requires up to 72 h, and goes no further than 75-80%, deglycosylated lutropin reaches greater than 90% conformational repair in less than 4 h. The efficacy of second-order absorption spectra in characterizing conformations and conformational changes in these molecules is demonstrated.

Animals↗
PubMed1984

Conformational comparison of human pituitary growth hormone and human chorionic somatomammotropin (human placental lactogen) by second-order absorption spectroscopy.

Second-order absorption spectra strongly suggest the presence of a hydrogen bond between the single Trp of human pituitary growth hormone (hGH) and a carboxylate ion. This hydrogen-bonded complex is buried within the hydrophobic interior of the hGH molecule. Although the homologous Trp in human chorionic somatomammotropin [human placental lactogen, HCS(hPL)] is also buried within the hydrophobic interior of the molecule, there is no evidence that it is hydrogen bonded in the native protein. However, during the early stages of thermolysin digestion of HCS(hPL), both difference and second-order absorption spectra do indicate the transient presence of a similar hydrogen-bonded Trp-carboxylate complex. The molar extinction coefficients of hGH and HCS(hPL) have been refined.

Growth Hormone↗
PubMed1983

Evidence for tertiary structure in aqueous solutions of human beta-endorphin as shown by difference absorption spectroscopy.

The presence of a distinct tertiary structure in aqueous solutions of human beta-endorphin has been demonstrated by difference absorption spectroscopy of thermolysin digests of the hormone and synthetic analogues. The results demonstrate that the alpha-amino group of Tyr1, Lys28, and some residue(s) between Thr6 and Ser10 are involved in forming and stabilizing the folded form of the molecule. Although a peptide corresponding to the first nine residues of human beta-endorphin shows definite evidence of tertiary structure, the pentapeptide methionine-enkephalin does not.

Amino Acid Sequence↗
PubMed1983

Isolation, purification, and characterization of deer mouse (Peromyscus maniculatus bairdii) prolactin.

Prolactin (PRL) secreted by Peromyscus maniculatus bairdii anterior pituitaries was purified by gel filtration on Sephadex G-100 and ion-exchange chromatography on DEAE-cellulose. Peromyscus PRL (pmPRL) eluted from Sephadex G-100 with an elution-to-void volume ratio of 1.9 and at a salt concentration of 100 mM NaCl on DEAE-cellulose. Electrophoretic homogeneity of the hormone was demonstrated in several gel systems. On 7 1/2% alkaline polyacrylamide gels, pmPRL migrated with an Rf of 0.65. The molecular weight of pmPRL was estimated at 24,000 to 26,000 by sodium dodecyl sulfate electrophoresis and molecular exclusion chromatography. The amino acid composition of pmPRL was similar to other mammalian PRLs including two tryptophan residues and three disulfide bonds. Leucine was found to be the NH2-terminal residue. Circular dichroism (CD) spectra indicated an alpha-helix content of 55 +/- 5%, a value typical for prolactin molecules. However, in the region of side-chain absorption, the CD spectrum displayed a weak, asymmetric, negative band near 299 nm, with the CD returning to slightly positive values at 292 nm. This CD pattern is not typical of other secreted or stored prolactins. In the pigeon crop-sac assay, pmPRL showed a prolactin-like activity of 21 IU/mg.

Amino Acids↗
PubMed1983

Conformational comparison of stored and secreted ovine pituitary prolactin.

Highly purified samples of stored and secreted ovine pituitary prolactin have been compared with regard to those conformational properties evidenced by ultraviolet absorption and circular dichroism measurements. No significant differences were found in any of the optical properties measured. The previously reported absence of tryptophanyl circular dichroism in the secreted forms of rat and mouse prolactins may be typical only of rodent hormones and not a general phenomenon.

Animals↗
PubMed1983

Studies on prolactin: conformational comparison of human, equine, and porcine pituitary prolactins.

The conformations of human, equine, and porcine pituitary prolactins, as evidenced by various optical properties, have been compared. The alpha-helix contents of all three proteins are essentially identical to each other (60 +/- 5%), as well as to prolactins isolated from other mammalian species. Direct absorption (zero and second-order), difference absorption, fluorescence emission, and circular dichroism spectra suggest that the majority of tyrosine and tryptophan side chains in these three proteins exist in very similar microenvironments within the folded forms of the hormones. Thus, the general conformational properties of these molecules are closely related to each other, and to other mammalian prolactins. Molar extinction and absorptivity values have been obtained at the absorption maximum of each species. In addition, a second molar extinction value has been determined at a particular wavelength found to be different for each, and which appears to be independent of the conformational state of the molecule. These absorptivities are useful in providing accurate prolactin concentrations in the 10(0) to 10(-2) mg/ml range. On incubation with the proteolytic enzyme thermolysin, all three hormones display an initial, short lag period during which little conformational change can be detected by difference absorption spectroscopy. For human and porcine prolactins, subsequent rates of proteolytically induced conformational collapse were found to be essentially identical. However, under similar conditions, equine prolactin loses its conformation significantly more slowly.

Animals↗
PubMed1982

Conformational studies of secreted mouse pituitary prolactin.

A secreted form of mouse pituitary prolactin has been shown to contain only a single tryptophan residue. All previously reported prolactins contain two tryptophans. Circular dichroism spectra indicate that secreted mouse prolactin is conformationally similar to stored forms of prolactin previously isolated from several other species, including its alpha-helix content (65%). However, like secreted rat prolactin, secreted mouse prolactin shows no tryptophan signal in its circular dichroism spectrum. All stored forms of prolactin studied to date display distinct tryptophan signals. This suggests the possibility that secretion of prolactin may be accompanied by modification of the protein's tertiary structure.

Animals↗