Simultaneous purification and polymerization method for bovine serum albumin preparation

Bovine serum albumin (BSA) has various applications in blood group serology and different research purposes. In this study purification of BSA has been compared with human serum albumin (HSA) using modified ethanol precipitation method based on the method of Cohn. The purification process was carried out under controlled conditions, particularly of ethanol concentration, pH, ionic strength and temperature. It was revealed that the produced BSA and HSA have purity more than 95%. It is obvious that HSA can be used, as a drug when the amount of its polymers is less than 5% whereas polymer generation is required in order to enhance the potentiating properties of BSA in agglutination of red cells. We propose here a simple and rapid two-step method for simultaneously purification and polymerization of BSA. By this method simply BSA with desired amount of polymers was obtained by 40% ethanol concentration.     

Presence of subunit structure in bovine follicle-stimulating hormone

Highly purified bovine follicle-stimulating hormone (FSH) potency 164 X NIH-FSH-S-1 by bioassay was estimated to be 210 +/- 4.2 X NIH-FSH-S-1 by radioligant-receptor assay using 125I-bovine FSH as tracer but only 120 +/- 1.8 X NIH-FSH-S-1 when 125I-human FSH tracer was used. The presence of subunit structure in bovine FSH was revealed by SDS-polyacrylamide gel electrophoresis. Furthermore, treatment of bovine FSH with 1 M propionic acid resulted in marked changes of mobilities upon polyacrylamide gel electrophoresis and elution profiles after gel filtration on Sephadex G-100 in addition to loss of biological activity by radioligand-receptor assay. After incubation, the propionic acid-treated bovine FSH recovered about 75% of the receptor-binding activity and regenerated protein bands of identical electrophoretic mobilities as the native hormone.     

Reliability of follicle-stimulating hormone measurements in serum

Background

Follicle-stimulating hormone (FSH), a member of gonadotropin family, is critical for follicular maturation and ovarian steroidogenesis. Serum FSH levels are known to fluctuate during different phases of menstrual cycle in premenopausal women, and increase considerably after the menopause as a result of ovarian function cessation. There is little existing evidence to guide researchers in estimating the reliability of serum FSH measurements. The objective of this study was to assess the reliability of FSH measurement using stored sera from an ongoing prospective cohort – the NYU Women's Health Study.

Methods

Sixty healthy women (16 premenopausal, 44 postmenopausal), who donated at least two blood samples at approximately 1-year intervals were studied. An immunoradiometric assay using a sandwich monoclonal antibodies technique was used to measure FSH levels in serum.

Results

The reliability of a single log-transformed FSH measurement, as determined by the intraclass correlation coefficient, was 0.70 for postmenopausal women (95% confidence interval (CI), 0.55–0.82) and 0.09 for premenopausal women (95% CI, 0–0.54).

Conclusions

These results suggest that a single measurement is sufficient to characterize the serum FSH level in postmenopausal women and could be a useful tool in epidemiological research. For premenopausal women, however, the reliability coefficient was low, suggesting that a single determination is insufficient to reliably estimate a woman's true average serum FSH level and repeated measurements are desirable.

     

Inactivation of viral agents in bovine serum by gamma irradiation

Cell culture origin or suckling mouse brain origin viruses of Akabane disease, Aino, bovine ephemeral fever, swine vesicular disease, hog cholera, bluetongue, and minute virus of mice were each suspended in bovine serum. Aliquots (1 mL) were exposed to various doses of gamma radiation from a 60Co source while at -68 degrees C. Aliquots (100-mL) of serum from a steer experimentally infected with foot-and-mouth disease virus were similarly irradiated. The samples were assayed for infectivity in cell culture systems before and after irradiation, and the data points were analyzed by linear regression. The irradiation doses (in megarads) necessary to inactivate one log10 of viral infectivity (D10) was calculated for each virus. D10 is otherwise known as the slope of the regression line. The r2 value, a measure of association with 1.0 = perfect fit, was also calculated for each regression line. The values (D10, r2) for each virus were as follows: Akabane, 0.25, 0.998; Aino, 0.35, 0.997; bovine ephemeral fever, 0.29, 0.961; swine vesicular disease, 0.50, 0.969; foot-and-mouth disease, 0.53, 0.978; hog cholera, 0.55, 0.974; bluetongue, 0.83, 0.958; and minute virus of mice, 1.07, 0.935.     

Interaction of ergosterol with bovine serum albumin and human serum albumin by spectroscopic analysis

This study was designed to examine the interactions of ergosterol with bovine serum albumin (BSA) and human serum albumin (HSA) under physiological conditions with the drug concentrations in the range of 2.99-105.88?μM and the concentration of proteins was fixed at 5.0?μM. The analysis of emission spectra quenching at different temperatures revealed that the quenching mechanism of HSA/BSA by ergosterol was the static quenching. The number of binding sites n and the binding constants K were obtained at various temperatures. The distance r between ergosterol and HSA/BSA was evaluated according to F?ster non-radioactive energy transfer theory. The results of synchronous fluorescence, 3D fluorescence, FT-IR, CD and UV-Vis absorption spectra showed that the conformations of HSA/BSA altered in the presence of ergosterol. The thermodynamic parameters, free energy change (ΔG), enthalpy change (ΔH) and entropy change (ΔS) for BSA-ergosterol and HSA-ergosterol systems were calculated by the van't Hoff equation and discussed. Besides, with the aid of three site markers (for example, phenylbutazone, ibuprofen and digitoxin), we have reported that ergosterol primarily binds to the tryptophan residues of BSA/HSA within site I (subdomain II A).     

Effect of gonadotropin-releasing hormone antagonists on serum follicle-stimulating hormone and luteinizing hormone under conditions of singular follicle-stimulating hormone secretion

Previous work has indicated that in long-term ovariectomized rats a potent antagonist to gonadotropin-releasing hormone (GnRH) suppressed serum luteinizing hormone (LH) more successfully than follicle-stimulating hormone (FSH). The present studies examined whether the rise in serum FSH which occurs acutely after ovariectomy, or during the proestrous secondary surge, depends on GnRH. In Experiment A, rats were ovariectomized at 0800 h of metestrus and injected with (Ac-dehydro-Pro1, pCl-D-Phe2, D-Trp3,6, NaMeLeu7)-GnRH (Antag-I) at 1200 h of the same day, or 2 or 5 days later. Antag-I blocked the LH response completely, but only partially suppressed serum FSH levels. Experiment B tested a higher dose of a more potent antagonist [( Ac-3-Pro1, pF-D-Phe2, D-Trp3,6]-GnRH; Antag-II) injected at the time of ovariectomy. The analog suppressed serum LH by 79% and FSH by 30%. Experiment C examined the effect of Antag-II on the day of proestrus on the spontaneous secondary surge of FSH, as well as on a secondary FSH surge which can be induced by exogenous LH. Antag-II, given at 1200 h proestrus, blocked ovulation and the LH surge expected at 1830 h, as well as increases in serum FSH which occur at 1830 h and at 0400 h. Exogenous LH triggered a rise in FSH in rats suppressed by Antag-II. In Experiment D proestrous rats were injected with Antag-II at 1200 h and ovariectomized at 1530 h. By 0400 h the antag had suppressed FSH in controls, but in the ovariectomized rats, a vigorous FSH response occurred.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational change of bovine serum albumin by heat treatment

The thermal denaturation of bovine serum albumin (BSA) was studied at pH 2.8 and 7.0 in the range of 2–65°C. The relative proportions of -helix, -structure, and disordered structure in the protein conformation were determined as a function of temperature, by the curve-fitting method of circular dichroism spectra. With the rise of temperature at pH 7.0, the proportion of -helix decreased above 30°C and those of -structure and disordered structure increased in the same temperature range. The structural change was reversible in the temperature range below 45°C. However, the structural change was partially reversible upon cooling to room temperature subsequent to heating at 65°C. On the other hand, the structural change of BSA at pH 2.3 was completely reversible in the temperature range of 2–65°C, probably because the interactions between domains and between subdomains might disappear due to the acid expansion. The secondary structure of disulfide bridges-cleaved BSA remained unchanged during the heat treatment up to 65°C at pH 2.8 and 7.0.     

Diffusion of bovine serum albumin in a neutral polymer solution

The diffusion coefficient D of bovine serum albumin through various solutions (pH 7.0, 0.5M NaCl) of polythylene oxide (M_w ～ 1 × 10⁵, 3 × 10⁵) was studied with quasielastic light scattering. In solutions of the 1 × 10⁵ polymer solution at polymer concentrations above 0.5 g/L, D is considerably greater than would have been expected from the viscosity of water:polymer mixtures, the deviations being larger at low protein concentration that at high protein concentration. With either polymer, D falls with increasing protein concentration.     

Urea-induced structural transformations in bovine serum albumin

Urea-induced unfolding of bovine serum albumin and one of its fragments containing domain II + III has been studied by difference spectral and fluorescence emission measurements. The unfolding-refolding curves of both the proteins showed the presence of at least one stable intermediate when the transition was monitored at 288 nm. The presence of the intermediate was not detectable at 293 nm where only tryptophan contributed towards the protein absorption. However, both the proteins did show the presence of intermediate when the denaturation was monitored fluorometrically. Since domain III of the albumin is devoid of tryptophan, it is concluded that the formation of intermediate in the unfolding-refolding transition of serum albumin involves (i) unfolding of domain III, (ii) minor structural transformations in domain II, and/or (iii) the separation of the sub-domains of domain III from each other.     

Radioimmunoassay of serum follicle-stimulating hormone and luteinizing hormone in the bottlenosed dolphin

Commercially available radioimmunoassay (RIA) kits for human follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were adapted for quantitation of these hormones in serum from bottlenosed dolphins (Tursiops truncatus). Serum samples from over 160 wild and 70 captive animals were assayed in order to determine basal concentrations of FSH and LH in these animals, as well as to detect possible differences between various groups. Mean FSH and LH levels for all animals were 0.22 +/- 0.08 and 0.37 +/- 0.18 ng/ml, respectively. Although wild animals had higher FSH and LH levels than captive ones, the differences were not statistically significant (P less than 0.07). However, both FSH and LH were significantly (P less than 0.01 and P less than 0.05, respectively) elevated in females when compared to males. Adults and peripubescent animals had significantly (P less than 0.01) higher LH levels than did juveniles. Among wild animals, serum concentrations of FSH and LH reflected seasonal differences. Samples obtained in early summer (Gulf of Mexico population) contained significantly (P less than 0.01) higher concentrations of FSH and LH than samples obtained in the fall (Indian River, Florida population). Both FSH and LH were significantly elevated in samples from confirmed pregnant animals as compared to the overall mean and to a sample from a confirmed nonpregnant female. Our observations indicate that these RIAs can reliably detect serum FSH and LH from bottlenosed dolphins and represent the first quantitation of these hormones in cetaceans.     

The binding of a thyroid hormone metabolite, 3-monoiodo-L-thyronine, to bovine serum albumin as measured by circular dichroism

The interaction between a thyroid hormone metabolite, 3-monoiodo-L-thyronine (3-T1) and bovine serum albumin (BSA) was investigated by using the CD method. An enhanced CD band was observed at the absorption wavelength region of 3-T1 around 293 nm suggesting the binding of 3-T1 to the BSA molecule. The ellipticity at 293 nm was measured at various molar ratios of 3-T1 to BSA, and the apparent binding constant and the maximum number of binding sites could be estimated as Kapp = 8.85 +/- 1.07 X 10(4) M-1 and n = 23.8 +/- 0.9 respectively. The CD of a mixture of BSA, 3-T1 and thyroxine (T4) was also studied at various pH's. The pH profile of the two characteristic CD bands at 293 nm and 320 nm, attributed to bound 3-T1 and T4, suggested that the optimum binding condition of 3-T1 was attained at alkaline pH of around 9, while that of T4 was attained over a wide pH range between 5-10. A significant role of the ionized 4'-hydroxyl group of 3-T1 in the binding reaction with BSA is also suggested.     

Inactivation by detergents of the proline transport system in membrane vesicles from Escherichia coli and its reactivation by bovine serum albumin

The proline transport system of membrane vesicles from Escherichia coli was inactivated by a low concentration of detergents such as deoxycholate, dodecyl sulfate and Triton X-100. The addition of a large amount of bovine serum albumin to membrane vesicles which had been treated with one of these detergents resulted in the restoration of the proline transport activity. The restoration of the transport activity by bovine serum albumin was most remarkable with the deoxycholate-inactivated membrane vesicle. 80% inactivation of the transport system with 0.005% deoxycholate was completely overcome by the addition of albumin. The degree of restoration was dependent on the concentration of albumin. Although albumin stimulated the proline transport activity itself, the stimulatory effect could not account for the restoration transport activity. The binding of deoxy[¹⁴C]cholate to the membrane vesicle was roughly proportional to the amount of detergent added. Deoxycholate once bound to the membrane vesicle was removed almost completely by the incubation with albumin. It is concluded that the removal of detergent from the membrane vesicle by bovine serum albumin results in the restoration of the proline transportactivity.