Activation of delta5-3-ketosteroid isomerase of bovine adrenal microsomes by serum albumins.

The Δ⁵-3-ketosteroid isomerase (EC 5.3.3.1) of bovine adrenal microsomes is activated as much as 10- to 20-fold by micromolar concentrations of bovine serum albumin. Comparable activations are observed with the serum albumins of 10 other mammalian species, but are not seen with ovalbumin or conalbumin. Evidence that the activation is attributable to the serum albumins, rather than to a small, firmly-bound ligand, is based on: (1) Failure to remove the stimulatory activity from the albumin by chloroform extraction, dialysis, or gel filtration; (2) Destruction of the activity by heating or by trypsin digestion; (3) Precipitation of the stimulatory activity of bovine serum albumin by specific antibody. Bovine serum albumin induces small decreases in the Michaelis constant for Δ⁵-androstene-3,17-dione, but most of the activational effect reflects an increase in the maximum velocity. Low concentrations of Triton X-100, which are without effect on the isomerase activity, prevent the activation by bovine serum albumin.     

Investigation of Ternary Complex Formations of Polyacrylic Acid with Bovine Serum Albumin in the Presence of Metal Ions by Fluorescence and Dynamic Light Scattering Measurements

In this paper, the complex formation of bovine serum albumin (BSA) and polyacrylic acid (PAA) in the presence metal ions at pH = 7 has been examined by using fluorescence and dynamic light scattering measurements. It has been observed that the most stable complexes of polyacrylic acid and bovine serum albumin have occurred in the presence of copper(II) ions. The other ions have the ability to form weak complexes between polyions and bovine serum albumin. To prior characterizing the interaction between bovine serum albumin and polyacrylic acid, the dynamic light scattering technique have been applied to determine the intensity-size distributions of the solutions of bovine serum albumin, polyacrylic acid, and ternary mixtures containing various molar ratios of bovine serum albumin to polyacrylic acid (the molar ratios of bovine serum albumin to polyacrylic acid has been taken equal to 0.5, 1.0, 1.5, 2.0 and 2.5) prepared at different molar ratios of copper(II) ions/acrylic acid unit. When the molar ratio of copper(II) ions to acrylic acid in the ternary mixtures has been lower than and equals to 0.3, two peaks have been observed in the curves of the intensity-size distributions due to contents of free bovine serum albumin and ternary complexes of polyacrylic acid-copper(II)-bovine serum albumin whereas when the molar ratio of copper(II) ions to acrylic acid equals to 0.4, the hydrodynamic diameter has pointed out only one peak. This result indicates that soluble and stable ternary complexes has occurred when the molar ratio of copper(II) ions to acrylic acid has been taken equal to 0.4.     

Protein hydration: A sucrose probe

The hydration of conalbumin, of myoglobin, of lysozyme, of carbon monoxide hemoglobin, of β-lactoglobin, of bovine serum albumin, of ovomucoid, of ribonuclease, and of egg albumin has been measured with equilibrium dialysis using sucrose as the probe at 30 °C. All proteins were at their isoelectric points except lysozymes and β-lactoglobulin and also samples of egg albumin which had been shifted to a more alkaline pH. Departure from their isoelectric points leads to an increase in the apparent protein hydration. Decreasing the temperature to 11.5 °C produces a slight increase in the hydration of egg albumin. A method is proposed for the calculation of protein hydration. The calculated protein hydration tends to be less than that determined experimentally for five of the proteins. There is satisfactory agreement with four of the proteins.     

Polymerization of Several Proteins by Ca2+-Independent Transglutaminase Derived from Microorganisms

α_s1-Casein and soybean globulins were polymerized and gelatinized by Ca²⁺-independent transglutaminase that was isolated from the culture filtrate of a microorganism thought to belong to Streptoverticillium sp. of actinomycetes. This enzyme polymerized such albumins as bovine serum albumin, human serum albumin and conalbumin in the presence of dithiothreitol. Rabbit myosin was polymerized by the present emzyme but actin was not. An RP-HPLC analysis after enzymic digestion of the polymerized a_sl -casein showed existence of the £-(y-Glu)Lys bond. Thus, it was confirmed that the polymerization was formed by a catalytic reaction of the transglutaminase.     

Partition of protein from binary mixtures by a batch foaming process

An experimental study to assess the partition of 3 binary protein mixtures when foam separated is presented (proteins considered were bovine serum albumin (BSA), conalbumin and lysozyme). Results show that selective partition of protein can be achieved and under certain conditions it is possible to strip the initial solution of BSA. However, purity of the foam phase is limited due to the non-selective carry-up of other proteins in the interstitial liquid.     

The Role of Histidine Residues in the Non-Enzyme Covalent Attachment of Glucose and Ascorbic Acid to Protein

Copper ions have been suggested to play a role in the non-covalent glycosylation (glycation) of proteins via transition metal-catalysed oxidations. We have further investigated “autoxidative glycosylation” by comparison of the behaviour of dog and bovine serum albumin with respect to the oxidative reactions of glucose and ascorbate. The proteins possess similar numbers of total amino residues available for glucose attachment but dog serum albumin contains fewer histidine groups and also lacks a high affinity copper-binding site. We find that the higher copper-binding capacity of bovine serum albumin is reflected in a lower rate of ascorbate oxidation as well as less protein oxidative damage than is the case for dog serum albumin. We also observe that modification of bovine serum albumin histidine groups by diethylpyrocarbonate enhances ascorbate-mediated protein fluorophore formation.     

Phosphorylation sites in riboflavin-binding protein characterized by fast atom bombardment mass spectrometry

The Lowry method for quantitation of protein was adapted to automated flow injection analysis. The procedure was developed using two different pure proteins: bovine serum albumin and hepatitis B surface antigen. The system was optimized for reagent concentration, pH, gain, temperature, sample volume, and output. The response of each protein was affected differently by temperature. The reaction slopes and absorbance values of the proteins were similar at 90 degrees C to allow quantitation of hepatitis surface antigen against bovine serum albumin. Advantages of the automated flow injection analysis Lowry procedure include: rapid analyses (90 samples/h), small sample volume (30 microliters, 100 microliters), fast response (20 s), reproducibility (less than or equal to 2% CV within an assay and 3 to 6% CV among assays), sensitivity (5 micrograms), and high correlation (99.8%) with manual assay. After a 30-min set-up period, the analyzer was available to assay protein on demand throughout the day, making it suitable for process and quality control testing.     

Interactions of proteins with immobilized metal ions: a comparative analysis using various isotherm models

Immobilized metal ion affinity chromatography (IMAC) is now a widely accepted technique for the purification of natural and recombinant therapeutic products and is beginning to find industrial applications. The design, optimization, and scale-up of a chromatographic process using IMAC demands a thorough understanding to be developed regarding the fundamental factors governing the various interactions between immobilized metal ions and proteins. Consequently, there is an immediate need to find out a theory that is able to account for these interactions most efficiently in a qualitative as well as a quantitative manner. In view of this requirement, the interactions of several model proteins (lysozyme, ovalbumin, bovine serum albumin, conalbumin, and wheat germ agglutinin) with metal (Cu(II), Ni(II))-chelated IDA (iminodiacetate) and tris(2-aminoethyl)amine were investigated. The adsorption data were analyzed using four isotherm models, viz., the general affinity interaction theory/Langmuir model, the Freundlich model, the Temkin model, and the Langmuir-Freundlich model, and the sorption parameters were computed. Although the first three models were applicable to some protein-IMA-M(II) systems, the Langmuir-Freundlich model appeared to be the most efficient model for explaining the interactions of proteins with IMA-M(II) gels. Also, this model was able to explain cooperativity and binding heterogeneity in quantitative terms. It is envisaged that this analysis would be useful in developing an improved understanding of protein-immobilized metal ion interactions and providing guidelines for designing preparative-scale separations using IMAC.     

Hemin-induced lysis of rat erythrocytes. Protective action of ceruloplasmin and different serum albumins

Hemin-induced lysis of rat erythrocytes is markedly reduced by ceruloplasmin (human) and serum albumins from different species, the order of effectiveness beings: bovine albumin approximately equal to ceruloplasmin greater than human albumin approximately equal to dog albumin greater than apotransferrin (human). Although the proteins studied had hemin binding capacity, the best protective agents, ceruloplasmin and bovine albumin, did bind hemin less strongly than human and dog albumin. The results suggest the existence of another protective mechanism, possibly involving an interaction between erythrocyte membranes and serum proteins.     

Changes in chlorophyll <Emphasis Type="Italic">a</Emphasis> and β-carotene fluorescence caused by hydrogen peroxide

Hydrogen peroxide is shown to change the fluorescence of β-carotene and chlorophyll a as well as various mixtures of the pigments in Langmuir films and in complex with bovine serum albumin in water solution. Similar effects were shown for zinc etioporphyrin II in the films. No shifts of the fluorescence peaks were registered in any case.     

Investigation of Bovine Serum Albumin (BSA) Attachment onto Self-Assembled Monolayers (SAMs) Using Combinatorial Quartz Crystal Microbalance with Dissipation (QCM-D) and Spectroscopic Ellipsometry (SE)

Understanding protein adsorption kinetics to surfaces is of importance for various environmental and biomedical applications. Adsorption of bovine serum albumin to various self-assembled monolayer surfaces including neutral and charged hydrophilic and hydrophobic surfaces was investigated using in-situ combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry. Adsorption of bovine serum albumin varied as a function of surface properties, bovine serum albumin concentration and pH value. Charged surfaces exhibited a greater quantity of bovine serum albumin adsorption, a larger bovine serum albumin layer thickness, and increased density of bovine serum albumin protein compared to neutral surfaces at neutral pH value. The quantity of adsorbed bovine serum albumin protein increased with increasing bovine serum albumin concentration. After equilibrium sorption was reached at pH 7.0, desorption of bovine serum albumin occurred when pH was lowered to 2.0, which is below the isoelectric point of bovine serum albumin. Our data provide further evidence that combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry is a sensitive analytical tool to evaluate attachment and detachment of adsorbed proteins in systems with environmental implications.     

Association equilibrium of d-methamphetamine and l-methamphetamine with serum albumin

The association equilibria for complex formation between serum albumin (bovine, rat) and the optical isomers of methamphetamine (MAMP) was determined using an ultrafiltration method. It was found that serum albumin/d-MAMP and serum albumin/l-MAMP complexes had distinctly different Scatchard plots with bovine and rat albumin. The binding parameters of each association equilibrium were estimated from the Scatchard plots by Rosenthal's graphic method. This distinguished two kinds of specific binding sites in terms of the association equilibrium between bovine serum albumin and d-MAMP, and one binding site for rat serum albumin and d-MAMP. One specific binding site was found between serum albumin and l-MAMP in both bovine and rat. Molar ellipticities, [θ], of peaks were decreased in the CD spectra of the complexes formed between bovine serum albumin and d-MAMP or l-MAMP when compared with the CD spectrum of bovine serum albumin alone. However, no difference in [θ] was found between the CD spectra of the enantiomers of MAMP in the measured wavelength range. The non-specific binding site was distinct from the specific binding site and resulting from altered tertiary structure of the albumin molecule. Chirality 10:742–746, 1998. © 1998 Wiley-Liss, Inc.     

Parameter scanning ultrafiltration: rapid optimisation of protein separation

High-resolution fractionation of proteins using ultrafiltration is feasible only at highly optimised conditions. Conventional process optimisation methodology demands both time and material. Pulsed sample injection ultrafiltration has been suggested as a rapid process optimisation technique. In the present work the scope of this technique is further extended by "parameter scanning ultrafiltration," which involves continuous change of a process parameter (e.g., pH, salt concentration). The time and material consumption are thus further reduced. The technique was validated using different proteins and membranes. Sieving coefficients at different pH and salt concentration were compared to those obtained in fixed parameter ultrafiltration experiments. As fractionation case studies the separation of monoclonal antibody from bovine serum albumin and separation of human IgG from human serum albumin were examined.     

Determination of coefficient of friction for self-expanding stent-grafts

Migration of stent-grafts (SGs) after endovascular aneurysm repair of abdominal aortic aneurysms is a serious complication that may require secondary intervention. Experimental, analytical, and computational studies have been carried out in the past to understand the factors responsible for migration. In an experimental setting, it can be very challenging to correctly capture and understand the interaction between a SG and an artery. Quantities such as coefficient of friction (COF) and contact pressures that characterize this interaction are difficult to measure using an experimental approach. This behavior can be investigated with good accuracy using finite element modeling. Although finite element models are able to incorporate frictional behavior of SGs, the absence of reliable values of coefficient of friction make these simulations unreliable. The aim of this paper is to demonstrate a method for determining the coefficients of friction of a self-expanding endovascular stent-graft. The methodology is demonstrated by considering three commercially available self-expanding SGs, labeled as A, B, and C. The SGs were compressed, expanded, and pulled out of polymeric cylinders of varying diameters and the pullout force was recorded in each case. The SG geometries were recreated using computer-aided design modeling and the entire experiment was simulated in ABAQUS 6.8/STANDARD. An optimization procedure was carried out for each SG oversize configuration to determine the COF that generated a frictional force corresponding to that measured in the experiment. The experimental pullout force and analytically determined COF for SGs A, B, and C were in the range of 6-9 N, 3-12 N, and 3-9 N and 0.08-0.16, 0.22-0.46, and 0.012-0.018, respectively. The computational model predicted COFs in the range of 0.00025-0.0055, 0.025-0.07, and 0.00025-0.006 for SGs A, B, and C, respectively. Our results suggest that for SGs A and B, which are exoskeleton based devices, the pullout forces increase upto a particular oversize beyond which they plateau, while pullout forces showed a continuous increase with oversize for SG C, which is an endoskeleton based device. The COF decreased with oversizing for both types of SGs. The proposed methodology will be useful for determining the COF between self-expanding stent-grafts from pullout tests on human arterial tissue.     

HPLC analysis of benzo[a]pyrene-albumin adducts in benzo[a]pyrene exposed rats. Detection of cis-tetrols arising from hydrolysis of adducts of anti- and syn-BPDE-III with proteins

Quantitation of protein-benzo[a]pyrene adducts represent a more sensitive analysis method than quantitation of benzo[a]pyrene-DNA adducts. By accurate analysis of benzo[a]pyrene-protein adducts several different molecular adduct forms can be studied. Male Wistar rats were injected i.p. with benzo[a]pyrene, and serum albumin was isolated and subjected to acid hydrolysis at 90 degrees C for 3 h. The hydrolysate was analyzed by HPLC with fluorescence detection. The HPLC profiles obtained after albumin hydrolysis from benzo[a]pyrene exposed animals were compared to similar HPLC profiles from in vitro adducted bovine serum albumin (BSA) and direct hydrolysis of both r-10,t-9-dihydrodiol-c-7,8-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (syn-BPDE-III) and r-10,t-9-t-dihydrodiol-t-7,8-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE-III). After acid hydrolysis of albumin from benzo[a]pyrene exposed rats, 6 fluorescent peaks were separated. Four of the peaks were isomers of benzo[a]pyrene-tetrahydrotetrols, (+/-)-benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol, (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol, (+/-)-benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol and (+/-)-benzo[a]pyrene-r-7,t-8,c-9,10-tetrahydrotetrol. In addition we found two fluorescent peaks, named X1 and X2 with retention times similar to the benzo[a]pyrene-tetrols. The unknown fluorescent peaks reacted similar to the four known tetrols in both dose response experiments and time course experiments. Fluorescent material with retention times equal to X1 and X2 were found after acid hydrolysis of syn-BPDE-III and anti-BPDE-III in acid and in hydrolysates from BSA treated in vitro with syn-BPDE-III and anti-BPDE-III. The ratio X1/X2 was relatively constant indicating epimerization equilibrium between these to species. Synchronous fluorescence analysis of fractions containing X1 or X2 from both in vivo and in vitro experiments showed fluorescence spectra characteristic of benzo[a]pyrene tetrols using a wavelength difference of 34 nm.     

Improved media for normal human muscle satellite cells: Serum-free clonal growth and enhanced growth with low serum

Summary We have developed a serum-free medium for clonal growth of normal human muscle satellite cells (HMSC). It consists of an optimized nutrient medium MCDB 120, plus a serum-free supplement, designated SF, that contains epidermal growth factor (EGF), insulin, dexamethasone, bovine serum albumin, and fetuin. Fibroblast growth factor was needed with dialyzed fetal bovine serum (dFBS) as the only other supplement, but in media containing SF, it was only slightly beneficial, and was omitted from the final medium without significant loss. Clonal growth of HMSC in MCDB 120 plus SF is as good as with 15% serum and 0.5% chicken embryo or bovine pituitary extract. However, growth is further improved by use of a doubly-supplemented (DS) medium containing both SF and 5% dFBS. Clonal growth of HMSC in the DS medium far exceeds that in previous media with any amount of serum, and monolayer growth is at least equal to that in conventional media with higher levels of serum. Cells grown in these media exhibit little differentiation, even when grown to high densities. However, they retain the capacity for extensive fusion and synthesis of increased creatine kinase when transferred to a serum-free differentiation-promoting medium, such as Dulbecco's modified Eagle's medium plus insulin. All experiments were done with clonal cultures of HMSC to insure that observed growth responses were always those of muscle cells. This research was supported by a grant from the Muscular, Dystrophy Association. Editor's statement This article describes the optimization of both the basal nutrient medium and growth factor requirements for human muscle cells in vitro. This system is critical for studies of normal muscle cell and molecular biology, as well as for understanding diseases of muscle such as Duchenne, Muscular Dystrophy.     

Development of sheep embryos in vitro in a medium supplemented with different batches of serum albumin

Variability in different lots of commercial serum albumin affects mammalian embryo development in culture. The composition of commercial preparations of ovine, bovine and defatted bovine serum albumin and a fraction of ovine serum containing proteins with a mean molecular weight of 65 kDa (fraction 3) was examined by polyacrylamide gel electrophoresis. All preparations were heavily contaminated with serum proteins other than albumin. Day-6 sheep morulae were cultured for 48 h in a basal bicarbonate-buffered salt solution supplemented with the commercial preparations of ovine, bovine or defatted bovine serum albumin. These three albumin preparations differed in their abilities to support the development of morulae into expanded blastocysts, but these differences disappeared when the basal medium was also supplemented with a component of ovine serum containing substances with molecular weights of less than 10 kDa. In the latter case, the three commercial albumin preparations and fraction 3 of ovine serum all supported full development in about 40-60% of morulae.     

不同pH值与电位耦合对牛血清白蛋白包被酒石酸长春瑞滨纳米粒制备工艺的影响

在反溶剂法制备纳米粒过程中,pH值在一定程度上会对其产生影响。本文通过在不同酸碱环境下运用反溶剂法制备牛血清白蛋白包被酒石酸长春瑞滨纳米粒,进而借助于电位耦合作用来研究纳米粒制备工艺。研究结果表明:当pH=4.5至7.5时,酒石酸长春瑞滨和牛血清白蛋白带有异种电荷,而当pH=2.5,3.5,8.5,9.5时它们均带有同种电荷。当pH=7.5时,牛血清白蛋白带有负电荷即-8.52 mV,酒石酸长春瑞滨带有正电荷即+4.48mV。此时得到牛血清白蛋白包被酒石酸长春瑞滨纳米粒粒径为193.3 nm,Zeta电位为-30.86 mV,而且在该pH下对纳米粒制备工艺进行了优化,最终它的载药量和包封率达到了45.6%和90.6%。     

Modulation of spermatozoa and zona pellucida properties by the soluble acrosome reaction-inducing factor of the ovulated egg-cumulus complex

Three sources of hamster periovulatory fluids (± heat inactivation at 56°C), with bovine serum albumin (BSA) as control, were tested for effects on penetration of three classes of eggs by hamster sperm precapacitated in BSA. These fluids were a soluble extract of cumulus oophorus fluid (COF) from the ovulated hamster egg-cumulus complex, serum, and follicular fluid. Egg types were ovulated, salt-stored (ovulated), and follicular. In both COF and serum, there were significant differences among egg types in mean penetration, and significant effects of fluid addition. In contrast, there was no effect of follicular fluid and no differences between follicular and stored eggs. For the follicular eggs (combined data, normalized, ranked), patterns of response to the three factors (± heating) were different: only unheated COF and heated serum increased penetration significantly above BSA control levels (average rank 20.2, 41.4, 38, for BSA, COF (unheated), serum (heated), respectively). This indicated that the active component in COF was heat labile, not present in either serum or follicular fluid, and, therefore, of oviductal origin. Oviduct and/or COF exposure of eggs and sperm was tested for effects as an acrosome reaction inducing factor (ARIF) for acrosome reactions (AR; zonabound and free-swimming sperm) and on sperm:zona binding and penetration. The COF ARIF for free-swimming sperm AR was heat stable. Penetration of follicular eggs increased after incubation in COF prior to sperm addition, but a greater response occurred when COF was added to eggs with sperm. In kinetic experiments, 25 min following sperm attachment, follicular eggs had lost 41% of initially bound sperm, vs. 23% for ovulated eggs, and had only 16 AR sperm/egg, vs. 26 for ovulated. Follicular eggs incubated in COF (then washed three times) had the same number of bound AR sperm as ovulated eggs. Acid solubilized zona pellucida (ASZP) from ovulated eggs was more effective as an ARIF per zona than ASZP from follicular eggs. Zonae of follicular eggs, as eivdenced by dissolution times in β-mercaptoethanol (β-MEOH), were not “harder” than those of ovulated eggs. There were differences in lectin binding antigens on zonae of both fresh and stored, follicular and ovulated, eggs. We conclude that multiple biological factors orchestrate sperm:egg interactions in the ampulla. Our data are consistent with the presence of at least three effective components: (1) the oviductal lectin-binding antigen (ZPO or oviductin), (2) an additional heat-labile component, and (3) the heat-stable ARIF for free-swimming sperm. © 1994 Wiley-Liss, Inc.     

The time-course development of the effects of ethanol ingestion on choline oxidase

Male rats were fed a low-fat diet containing 36% of calories as ethanol, and the time-course development of the effects of ethanol on liver mitochondrial oxidation of choline was determined. Ethanol induced an increase in choline oxidase at days 2, 5 and 7 after being introduced into the diet. Due to an observed 32% increase in total fatty acids in the whole liver, defatted bovine serum albumin was added to the buffer used to homogenize the liver. The presence of bovine serum albumim resulted in a significant decrease in choline oxidase activity at days 2 and 5; however, ethanol still induced an increase in choline oxidase activity in these mitochondria. The total fatty acid concentration of mitochondria prepared in the absence of bovine serum albumin increased steadily until day 5; however, by day 7 the fatty acid concentration had returned to control levels. The addition of bovine serum albumin to the homogenization medium prevented the increase in the total amount of fatty acids. The fatty acid composition of the bovine serum albumin-treated mitochondria, however, was not different from the mitochondria is isolated in the absence of bovine serum albumin. Further, the addition of a free fatty acid to isolated mitochondrial preparations caused about a 100% increase in choline oxidase. These data are consistent with the idea that choline oxidase may be regulated to some extent by an influx or an increase in free fatty acids in the liver as a result of ethanol ingestion. Thus, a second mechanism has been described which contributes to the increase in choline oxidase after ethanol ingestion.