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.

A turbidimetric method to determine visual appearance of protein solutions.

An instrumental method to analyze protein solutions for visual appearance is described which is based on spectrophotometric comparison to reference suspensions with varying degrees of turbidity. This method provides a useful substitute for visual inspection of uniform opalescent suspensions in that it is more convenient and less time-consuming and has the potential to be more reproducible, accurate and objective. Established categories of opalescence based on European Pharmacopoeial reference suspensions were determined using turbidity measured as optical density in the 340-360 nm range. A comparison of the mean optical density of a protein sample to those of the reference suspensions allowed a more reproducible assignment of the sample's category of opalescence than that of visual inspection.

Drug dependence with oestrogen replacement therapy.

Dependence on some drugs can be hard to recognize. Hormone replacement therapy (HRT) has been widely prescribed only in the past two decades, and the indications for treatment and the risk/benefit ratio are still disputed. Oestrogens are psychoactive: they lift mood, can be given by injection, and their use has powerful psychological effects. Reports of women with supraphysiological oestradiol concentrations may represent tolerance and withdrawal. Dependence on substances occurring naturally in the body has been reported before. We propose that HRT dependence occurs.

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.

In-patient treatment for drug abuse.

During 1984 there were 253 admissions to the in-patient drug dependence treatment unit at Tooting Bec Hospital. Of the 198 patients responsible for these admissions, 60% were male and 43% were over the age of 30 years. There were 151 patients admitted for opiate detoxification, and 75% completed the withdrawal schedule. However, only 15 out of 25 patients admitted for benzodiazepine withdrawals were found to be physically dependent. Other reasons for admission included stabilisation of the dose of opiate (24%) and the treatment of physical complications of addiction.

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.

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.

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.

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.

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%.

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.