The effect of serum albumin and the effect of cell concentration on the in vitro growth of mouse and rat lymphocytes.

The growth of mouse and rat T and B lymphocytes, activated by concanavalin A or lipopolysaccharide, is increased over growth in protein-free medium 5–20-fold by human or bovine serum albumin. The growth is dependent on the concentration of albumin. At a concentration of 2–4 mg/ml the growth rate is the same as in the presence of 10% fetal bovine serum. Of the other serum proteins (Cohn fractions) only human fraction VI supports growth somewhat while human fractions II–IV and bovine fraction VI do not support growth. The growth of mouse and rat lymphocytes is greatly suppressed if lymphocytes are cultured at high cell concentrations, and the growth-promoting ability of serum albumin cannot be detected under such conditions. The growth rate can be improved by daily adjustment of the pH, by daily refeeding, and by daily change of medium. The growth inhibitory activity can be removed largely by dialysis. It is concluded that the suppression of growth at high cell concentrations is caused by a combination of effects, i.e., a shift of pH, lack of nutrients, and accumulation of cellular metabolites.     

Intermolecular interactions in solutions of serum albumin

The mechanisms of intermolecular protein complex formation were studied by the example of monomers, oligomers and aggregates of bovine serum albumin (BSA) depending on the protein concentration, pH and urea concentration. Using dynamic light scattering (DLS), analytical ultracentrifugation (AUC) and PAG electrophoresis we have shown the existence of dynamic equilibrium between monomers and aggregates in BSA solution. Decreasing pH of the solution (4.0–1.0) resulted in increasing sizes of the aggregates. In the solutions with low urea concentrations (below 2 M) the sizes of aggregates decreased, while higher urea concentrations (2–8 M) induced formation of larger aggregates due to the unfolding of the protein.     

Urea denaturation of bovine serum albumin at pH 9

The action of 5 m urea on bovine serum albumin has been studied at pH 9.0 and 25°C. Analysis by the acrylamide gel electrophoresis revealed the presence of a few components 1, 1′, 2, 3, 4 and 5. The components 1 and 1′ are monomers, component 2 is a dimer, and components 3, 4 and 5 are aggregates. In presence of SH blocking reagent, bovine serum albumin gave only the zone 1, indicating that the components 1′-5 were formed by the SH to S-S exchange reactions. Component 1′ was formed by the intramolecular SH to S-S exchange reaction, and components 2–5 were formed by the intermolecular exchange reaction. Addition of cysteine either to bovine serum albumin or to the SH-blocked bovine serum albumin increased the percent of zone 1′, indicating that a complex bovine serum albumin-cysteine was formed or that the SH-catalyzed structural alteration occurred in bovine serum albumin. Components 1, 1′, 2 and 3 were isolated separately by the preparative disc gel electrophoresis. The sedimentation coefficients 1 and 1′ differed slightly indicating that they were different monomers, and values were slightly smaller than the normal value of bovine serum albumin, indicating that these components were in slightly expanded state. Isolated component 1 was exposed to 5 m urea again, but no further change occurred. This supports the concept of microheterogeneity of bovine serum albumin.     

Production of recombinant human serum albumin from Saccharomyces cerevisiae

Human serum albumin has been constitutively expressed in a Saccharomyces cerevisiae brewing yeast. After cell growth and disruption the product was associated with the insoluble fraction and represented approximately 1% of total cell protein. After the cell debris was extensively washed, the albumin was solubilized with 8 M urea and 28 mM 2-mercaptoethanol in 50 mM sodium carbonate buffer, pH 10. The denatured albumin was refolded by dialysis and further purified by anion exchange and gel filtration chromatography. Losses of renatured material could be reduced, or higher protein concentrations used during refolding, if the denatured product was purified by cation-exchange chromatography in urea prior to refolding. Apart from an additional N-terminal N-acetyl methionine, the refolded product proved identical to human serum albumin derived from plasma when compared by a variety of physical, chemical, and biological analytical methods.     

Domain II + III of bovine serum albumin: Isolation and its characterization

Some properties of a fragment of bovine serum albumin containing residues 184–582 of the protein sequence, produced by cyanogen bromide cleavage, have been reported. Urea-induced difference spectra of the fragment showed considerable exposure of aromatic chromophores by 8 M urea. Reversible unfolding of the fragment by urea, as followed by difference spectral measurements at 30°C, pH 7.0, occurred in two distinct steps involving at least 3 major conformational states, namely the native (N), intermediate (X) and completely denatured (D) states. The co-operativity values for the two transitions, N⇌X and X⇌Dwere found to be 4.0 and 16.4, respectively. Analysis of the data on bilirubin binding to bovine serum albumin and its fragment suggested that the fragment retains significant amount of its native structure. However, hydrodynamic parameters such as Stokes radius (3.f14 nm), diffusion coefficient (6.98 × 10⁻⁷cm²/s) and frictional ratio (1.32) obtained by analytical gel chromatography as well as intrinsic viscosity (4.31 ml/g) indicates some asymmetry in the fragment molecule.     

Formation of a molten globule like state in bovine serum albumin at alkaline pH

Little work has been done to understand the folding profiles of multi-domain proteins at alkaline conditions. We have found the formation of a molten globule-like state in bovine serum albumin at pH 11.2 with the help of spectroscopic techniques; like far and near ultra-violet circular dichroism, intrinsic and extrinsic fluorescence spectroscopy. Interestingly, this state has features similar to the acid-denatured state of human serum albumin at pH 2.0 reported by Muzammil et al. (Eur J Biochem 266:26–32, 1999). This state has also shown significant increase in 8-anilino-1-naphthalene-sulfonate (ANS) binding in compare to the native state. At pH 13.0, the protein seems to acquire a state very close to 6 M guanidinium hydrochloride (GuHCl) denatured one. But, reversibility study shows it can regain nearly 40% of its native secondary structure. On the contrary, tertiary contacts have disrupted irreversibly. It seems, withdrawal of electrostatic repulsion leave room for local interactions, but disrupted tertiary contacts fail to regain their original states.     

Spectroscopic studies on the complex formation of suramin with bovine and human serum albumin.

The binding of suramin to bovine and human serum albumin was investigated by gel filtration and spectroscopic measurements. Besides some low-affinity binding sites suramin has, on the bovine serum albumin molecule one and on the human serum albumin molecule two, high-affinity binding sites. Spectroscopic measurements reveal that there are large differences between the albumins in the mechanism of binding to the high-affinity binding sites. Further, it is suggested that high concentrations of suramin provoke an unfolding of the albumin moleculse. In order to explain the unusual behaviour of suramin in connection with the displacement of other ligands from the albumin binding the fluorescence probe 1-anilino-8-naphthalenesulfonic acid (ANS) was employed as a reporter group molecule for fluorescence as well as circular dichroism measurements. By these measurements it could be shown that suramin greatly influences the microorganization of both albumin molecules. In the case of these measurements large differences between bovine and human serum albumin were also found.     

Conformation inversion of bilirubin formed by reduction of the biliverdin-human serum albumin complex: evidence from circular dichroism.

As shown by circular dichroism spectroscopy, biliverdin preferentially adopts an M-helicity conformation on human serum albumin in aqueous buffer, pH 7.5, whereas biliverdin exhibits only a weak preference for the P-helicity conformation on bovine serum albumin at the same pH. Upon rapid reduction of the complexes with sodium borohydride, P-helicity bilirubin-IX alpha is obtained on the human albumin complex, and M-helicity bilirubin-IX alpha is obtained on the bovine serum albumin complex. Thus, biliverdin in effect undergoes an inversion of chirality upon reduction. Since the reduction did not afford a rubin with the same helicity as that of the verdin, the observations point to a hitherto undetected conformational mobility of albumin-bound bilirubin.     

Salt-induced refolding in different domains of partially folded bovine serum albumin

In our earlier communication on urea denaturation of bovine serum albumin (BSA), we showed significant unfolding of domain III along with domain I prior to intermediate formation around 4.6-5.2 M urea based on the binding results of domain specific ligands:chloroform, bilirubin and diazepam for domains I, II and III, respectively. Here, we present our results on the salt-induced refolding of the two partially folded states of BSA obtained at 4.5 M urea and at pH 3.5, respectively. Both these states were characterized by significant unfolding of both domains I and III as indicated by decreased binding of chloroform and diazepam, respectively. Salt-induced stabilization of partially folded states of BSA was accompanied by nearly complete refolding of both domains I and III as the binding isotherms of chloroform and diazepam obtained in presence of approximately 1.0 M KCl were nearly identical to that obtained with native BSA at pH 7.4. From these observations, it can be concluded that the anion binding sites on serum albumin are not only confined to domain III (C-terminal region) but few sites are also present on domain I (or N-terminal region) of the protein.     

Molten globule intermediates of human serum albumin in low concentration of urea

Interaction of non-electrolytes such as urea with proteins especially at lower concentrations is opening-up newer concepts in the understanding of protein stability and folding in proteomics. In this study, the secondary and tertiary structural characteristics and thermal stability of human serum albumin at lower concentrations of urea have been monitored. The protein attains a molten globule like structure at concentration urea below 2 M. This structural state also shows an increase in the alpha-helical content as compared to the native state. At concentrations of urea above 2 M, human serum albumin starts unfolding, resulting in a three-state transition with two mid points of transitions at around 4 M and 7 M urea concentrations. The characteristics of the partially folded intermediates are discussed with respect to the three component system analyses. Preferential hydration dominates over preferential interaction at lower concentration of urea (up to 2.5 M) and at higher concentration, the preferential interaction overtakes preferential hydration in a competitive manner. Formation of structural intermediates at lower concentration of urea is hypothesized as a general phenomenon in proteins and fits in with the observation with preferential interaction parameters by Timasheff and co-workers in the case of lysozyme and ribonuclease at different pH values.     

Effect of physiological concentration of urea on the conformation of human serum albumin

We report that the presence of very low concentrations (<0.1 M) of urea, a widely used chemical denaturant, induces structure formation in the water-soluble globular protein human serum albumin (HSA) at pH 7. We have presented results suggesting an almost 8% and 5% increase in alpha-helix in the presence of 10 mM urea (U) and 20 mM monomethylurea (MMU), respectively. Far and near-UV circular dichroism studies along with tryptophan fluorescence and 1-anilino-8-naphthalenesulphonicacid (ANS) binding support our view. We hypothesize that both U and MMU, at such low concentrations, modify the solvent structure, increase the dielectric constant and consequently increase hydrophobic forces resulting in enhanced alpha-helical content. The implications of these results of the lower urea regime are significant because the physiological blood urea ranges from 2.5 to 7.5 mM.     

Elucidating the mode of action of urea on mammalian serum albumins and protective effect of sodium dodecyl sulfate

The effect of sodium dodecyl sulfate (SDS) on human, bovine, porcine, rabbit and sheep serum albumins were investigated at pH 3.5 by using various spectroscopic techniques like circular dichroism (CD), intrinsic fluorescence and dynamic light scattering (DLS). In the presence of 4.0 mM SDS the secondary structure of all the albumins were not affected as measured by CD but fluorescence spectra revealed 8.0 nm blue shift in emission maxima. We further checked the stability of albumins in the absence and presence of 4.0 mM SDS by urea and temperature at pH 3.5. In the absence of SDS, urea starts unfolding both secondary as well as tertiary structural elements of the all the albumins at ∼2.0 M urea but in the presence of 4.0 mM SDS, urea was unable to unfold even up to 9.0 M. The albumins were thermally less stable at pH 3.5 with decrease in T_m but in the presence of 4.0 mM SDS, the T_m was increased. From this study, it was concluded that SDS is showing a protective effect against urea as well as thermal denaturation of albumins. This behavior may be due to electrostatic as well as the hydrophobic interaction of SDS with albumins. Further, we have proposed the mechanism of action of urea. It was found that urea interacted with proteins directly when proteins are in charged form. Indirect interaction may be taking place when the environment is more hydrophobic.     

Urea-induced denaturation of human serum albumin labeled with acrylodan

We induced the denaturation of unlabeled human serum albumin (HSA) and of similar albumin labeled with acrylodan (6-acryloyl-2-dimethylamino naphthalene) with urea and studied the transition profiles using circular dichroism and fluorescence spectroscopy. The circular dichroism spectra for both albumin preparations resulted in the same curves, thus indicating that labeling with acrylodan does not perturb the conformation of HSA. Our results indicate that the denaturation of both albumin preparations takes place at a single, two-state transition with midpoint at about 6 M urea, due to the unfolding of its domain II. It is important to point out that even at 8 M urea, some residual structure remains in the HSA. Great changes in the fluorescence of the dye bound to the protein were observed by addition of solid guanidine hydrochloride to the protein labeled with acrylodan dissolved in 8 M urea, indicating that domain I of this protein was not denatured by urea.     

Bactericidal effect of fatty acids on mycobacteria, with particular reference to the suggested mechanism of intracellular killing

The effect of fatty acids on Mycobacterium smegmatis was examined in vitro at pH 5.0 to 7.0 to determine the role of fatty acids in the intracellular killing of mycobacteria. Unsaturated fatty acids showed strong bactericidal activity in low concentrations (0.005 to 0.02 mM), whereas saturated fatty acids, except for lauric and myristic acids, were not very effective even at a concentration of 0.2 mM. Addition of a saturated fatty acid (palmitic or stearic acid) to an unsaturated fatty acid (oleic or linoleic acid) did not strongly interfere with the bactericidal effect of the unsaturated fatty acid at pH 5.0 and 6.0. Ca2+ (3.0 mM), Mg2+ (1.0 mM), and gamma-globulin (0.4%) showed weak reversal effects on the bactericidal activity of unsaturated fatty acids at pH 5.0 and 6.0. Serum albumin and serum showed strong reversal effects. The concentrations of each fatty acid in a mixture (molar ratio, 1:1:1:1) of oleic, linoleic, palmitic, and stearic acids required for the killing of M. smegmatis in the presence of 2% serum (bovine, rabbit, or human) were 0.05 to 0.10 mM at pH 5.0 and 6.0 and 0.05 to 0.20 mM at pH 7.0, depending on the serum used. The susceptibilities of M. kansasii, M. bovis strain BCG, and M. tuberculosis to the mixture of the four fatty acids in the presence of 2% bovine serum were similar to that of M. smegmatis, although M. fortuitum was more resistant.     

Photo-induced riboflavin binding to the tryptophan residues of bovine and human serum albumins

Summary The photobinding between riboflavin and the Trp residues from human and bovine serum albumins at two pH-dependent protein conformations was studied. At pH 7.0 both proteins showed photo-adduct formation with hyperbolic kinetics. In the bovine serum albumin this is attributed to the different locations of the two Trp residues. In the case of the human serum albumin, which has only one Trp residue, this behaviour may be related to different molecular conformations of the protein, as is also manifest in the iodide quenching experiments. At pH 3.5, the kinetics of the photo-adduct formation were found to be slower and showed a monophasic behaviour. These results are due to the conformational change of these proteins at acidic pH; the Trp residues of both proteins being now located in a more hydrophobic environment. When bovine serum albumin was anaerobically irradiated at pH 7.0 in the presence of¹⁴C-riboflavin and then cleaved by CNBr, two peptides were obtained, containing the Trp-134 and Trp-212 residues, respectively. The incorporation of¹⁴C-riboflavin in these samples was significantly higher at the level of the peptide containing the Trp-134 residue. Furthermore, it was demonstrated, that the energy transfer from enzymatically generated triplet acetone to riboflavin can also promote the binding of this vitamin to the Trp residues of human and bovine serum albumins.     

Purification and Characterization of an Extracellular Acid Proteinase from the Ectomycorrhizal Fungus Hebeloma crustuliniforme

Hebeloma crustuliniforme produced an extracellular acid proteinase in a liquid medium containing bovine serum albumin as the sole nitrogen source. The proteinase was purified 26-fold with 20% activity recovery and was shown to have a molecular weight of 37,800 (as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and an isoelectric point of 4.8 +/- 0.2. The enzyme was most active at 50 degrees C and pH 2.5 against bovine serum albumin and was stable in the absence of substrates at temperatures up to 45 degrees C and pHs between 2.0 and 5.0. Pepstatin A, diazoacetyl-dl-norleucine methylester, metallic ions Fe and Fe, and phenolic acids severely inhibited the enzyme activity, while antipain, leupeptin, N-alpha-p-tosyl-l-lysine chloromethyl ketone, and trypsin inhibitor inhibited the activity moderately. The proteinase hydrolyzed bovine serum albumin and cytochrome c rapidly compared with casein and azocasein but failed to hydrolyze any of the low-molecular-weight peptide derivatives tested.     

Heparin-binding properties of human serum spreading factor

Human serum spreading factor (SF) is a blood glycoprotein that promotes attachment and spreading and influences growth, migration, and differentiation of a variety of animal cells in culture. SF purified from human plasma or serum by chromatographic methods reported previously (Barnes, D. W., and Silnutzer, J. (1983) J. Biol. Chem. 258, 12548-12552) does not bind to heparin-Sepharose under conditions of physiological ionic strength and pH. In a further examination of the heparin-binding properties of human serum SF, we found that exposure of purified SF to 8 M urea altered several properties of the protein, including heparin affinity, and these alterations remained after removal of the urea from SF solutions. Urea-treated SF bound to heparin under physiological conditions, and salt concentrations of 0.4 M or higher were required for elution of urea-treated SF from heparin-Sepharose at pH 7.0. The alteration of heparin-binding properties of SF also was observed upon exposure of the protein to heat or acid. Treatment of SF with urea, heat, or acid resulted additionally in greatly decreased cell spreading-promoting activity of the molecule. The decreased biological activity was associated with a reduced ability of the treated SF to bind to the cell culture substratum, a prerequisite for the attachment-promoting activity of the molecule. Experiments examining the heparin-binding properties of native SF in unfractionated human plasma indicated that the major portion of SF in blood did not bind to heparin under conditions of physiological ionic strength and pH.     

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.     

Frontal gel chromatographic analysis of the interaction of a protein with self-associating ligands: aberrant saturation in the binding of flavins to bovine serum albumin

Frontal gel chromatography is an accurate method to obtain the total free ligand concentration of a protein-ligand mixture in which ligands self-associate. The average number of bound ligands per protein molecule is obtained as a function of the total free ligand concentration. The method was applied to the interaction of bovine serum albumin with self-associating flavins. The binding curves for FMN and FAD leveled off at about 0.7 and 0.5, respectively. These data were simulated well by a binding model where flavins undergo isodesmic indefinite self-association and the monomer alone binds to a single binding site of albumin. The isodesmic association constants of FMN and FAD were (1.7 +/- 0.1) x 10(2) and (2.2 +/- 0.3) x 10(2) M(-1), respectively. The binding constants of the monomer of FMN and FAD were (7.6 +/- 0.2) x 10(2) and (3.5 +/- 0.2) x 10(2) M(-1), respectively. FMN competitively inhibited the binding of FAD to albumin. The affinity to flavins was in the following order at pH 5.8: lumiflavin, FMN, riboflavin, and FAD. The SH modification and the binding of palmitate did not affect the FMN binding to bovine albumin at pH 5.8. As pH increased from 5.8 to 9.0, the affinity to FMN of bovine albumin decreased 3-fold, whereas that of human albumin increased about 80-fold. The present study clearly showed how isodesmic self-association of a ligand can cause apparent saturation of the interaction of a protein with the ligand at levels lower than 1.     

Studies on the growth-stimulatory activity of pigeon milk-comparison and synergistic effects with serum

Pigeon milk, a nutritive secretion from the crop of breeding pigeons, was tested (on v/v basis) for growth factor activity either separately or in combination with other growth supplements. Synthesis of DNA in confluent monolayers of quiescent Chinese hamster ovary cells was enhanced by the homogenates of pigeon milk in the presence of both fetal bovine serum and bovine serum albumin, although the response with fetal bovine serum was greater than that with bovine serum albumin. The in vitro growth stimulation by pigeon milk was also reflected in the increase in cell number. Specific activity of pigeon milk growth factor, measured against both Chinese hamster ovary cells and mouse embryo fibroblasts, was found to be higher than that of fetal calf serum, fetal bovine serum, and goat, horse, pig and human serum. The growth-stimulatory property of pigeon milk did not change in the first 5 days of its secretion.Abbreviations BSA bovine serum albumin - CHO Chinese hamster ovary cells - DMEM Dulbecco's modified minimum essential medium - DNA deoxyribonucleic acid - EDTA ethylenediaminetetraacetic acid - EGF epidermal growth factor - FBS fetal bovine serum - FCS fetal calf serum - GF growth factor - GS goat serum - NIH/3T3 mouse embryo fibroblasts - PBS phosphate-buffered saline - PDGF platelet-derived growth factor - PM pigeon milk