Polysaccharides as Modulators of Bovine Serum Albumin Fibril Formation

Alzheimer's disease (AD) involves amyloid beta (Abeta) accumulation, oxidative damage, and inflammation, and risk is reduced with increased antioxidant and anti-inflammatory consumption. The phenolic yellow curry pigment curcumin has potent anti-inflammatory and antioxidant activities and can suppress oxidative damage, inflammation, cognitive deficits, and amyloid accumulation. Since the molecular structure of curcumin suggested potential Abeta binding, we investigated whether its efficacy in AD models could be explained by effects on Abeta aggregation. Under aggregating conditions in vitro, curcumin inhibited aggregation (IC(50) = 0.8 microM) as well as disaggregated fibrillar Abeta40 (IC(50) = 1 microM), indicating favorable stoichiometry for inhibition. Curcumin was a better Abeta40 aggregation inhibitor than ibuprofen and naproxen, and prevented Abeta42 oligomer formation and toxicity between 0.1 and 1.0 microM. Under EM, curcumin decreased dose dependently Abeta fibril formation beginning with 0.125 microM. The effects of curcumin did not depend on Abeta sequence but on fibril-related conformation. AD and Tg2576 mice brain sections incubated with curcumin revealed preferential labeling of amyloid plaques. In vivo studies showed that curcumin injected peripherally into aged Tg mice crossed the blood-brain barrier and bound plaques. When fed to aged Tg2576 mice with advanced amyloid accumulation, curcumin labeled plaques and reduced amyloid levels and plaque burden. Hence, curcumin directly binds small beta-amyloid species to block aggregation and fibril formation in vitro and in vivo. These data suggest that low dose curcumin effectively disaggregates Abeta as well as prevents fibril and oligomer formation, supporting the rationale for curcumin use in clinical trials preventing or treating AD.

Bovine serum albumin (BSA) containing buffers are the standard blocking buffer in biosensing, yet human serum is the intended application for most clinical sensors. However, the effect of human serum albumin (HSA) on binding assays remains underexplored. A simple and well-studied assay (human IgG/goat anti-human IgG) was investigated with a surface plasmon resonance (SPR) sensor to address this fundamental question in sensing. Calibrations were performed with buffers containing various concentrations of bovine or human serum albumin, as well as full and diluted bovine or IgG-depleted human serum. It was found that HSA or human serum, but not BSA or bovine serum, significantly affected the SPR shift and binding constants of the assay. Interestingly, large differences were also observed depending on whether the animal or human antibody was immobilized on the SPR chip for detection, highlighting that matrix protein/analyte/receptor interactions play a significant role in the response. We find that the interaction of soluble HSA with human IgG interferes with the recognition region, affecting the binding constant, and thus results obtained in BSA are not necessarily applicable to clinical samples or in vivo conditions. We also clearly demonstrate why a minimum dilution of 1 : 10 is often required in SPR assays to remove most background effects. Taken together, these results show that: (1) BSA does not affect the binding constant between antibodies and thus serves its purpose well when only surface blocking is intended, (2) HSA is an adequate surrogate for human serum in assay optimization, and (3) blocking buffers should be prepared with HSA in the optimization steps of assays to be translated to human blood or serum.

Childhood Membranous Nephropathy and Dietary Antigens

During antibiotic drug development, media are frequently spiked with either serum/plasma or protein supplements to evaluate the effect of protein binding. Usually, previously reported serum or plasma protein binding values are applied in the analysis. The aim of this study was to evaluate this approach by experimentally measuring free, unbound concentrations for antibiotics with reportedly high protein binding and their corresponding antimicrobial activities in media containing commonly used protein supplements. Free, unbound ceftriaxone and ertapenem concentrations were determined in bacterial growth medium with and without bovine/human serum albumin, as well as adult bovine serum and human plasma using in vitro microdialysis. The corresponding antimicrobial activity was determined in MIC and time-kill curve experiments using Escherichia coli ATCC 25922 and Streptococcus pneumoniae ATCC 6303 as test strains. A semimechanistic maximum effect model was simultaneously fitted to the data and respective EC(50) (concentration at half-maximum effect) values compared. Protein binding differed significantly for ceftriaxone (P < 0.05) between human plasma (76.8 +/- 11.0%) and commercially available bovine (20.2 +/- 8.3%) or human serum albumin (56.9 +/- 16.6%). Similar results were obtained for ertapenem (human plasma, 73.8 +/- 11.6%; bovine serum albumin, 12.4 +/- 4.8%; human serum albumin, 17.8 +/- 11.5%). The MICs and EC(50)s of both strains were significantly increased (P < 0.05) for ceftriaxone when comparing human and bovine serum albumin, whereas the EC(50)s were not significantly different for ertapenem. Free, unbound antibiotic concentrations differed substantially between plasma and protein supplements and correlated well with antimicrobial efficacy. Therefore, free, active concentrations should be measured in the test system instead of correcting for literature protein binding values.

Per- and polyfluoroalkyl substances (PFAS) are a class of over 8,000 chemicals that are persistent, bioaccumulative, and toxic to humans, livestock, and wildlife. Serum protein binding affinity is instrumental in understanding PFAS toxicity, yet experimental binding data is limited to only a few PFAS congeners. Previously, we demonstrated the usefulness of a high-throughput, in vitro differential scanning fluorimetry assay for determination of relative binding affinities of human serum albumin for 24 PFAS congeners from 6 chemical classes. In the current study, we used this differential scanning fluorimetry assay to comparatively examine differences in human, bovine, porcine, and rat serum albumin binding of 8 structurally informative PFAS congeners from 5 chemical classes. With the exception of the fluorotelomer alcohol 1H,1H,2H,2H-perfluorooctanol (6:2 FTOH), each PFAS congener bound by human serum albumin was also bound by bovine, porcine, and rat serum albumin. The critical role of the charged functional headgroup in albumin binding was supported by the inability of serum albumin of each species tested to bind 6:2 FTOH. Significant interspecies differences in serum albumin binding affinities were identified for each of the bound PFAS congeners. Relative to human albumin, perfluoroalkyl carboxylic and sulfonic acids were bound with greater affinity by porcine and rat serum albumin, and perfluoroalkyl ether congeners bound with lower affinity to porcine and bovine serum albumin. These comparative affinity data for PFAS binding by serum albumin from human, experimental model and livestock species reduce critical interspecies uncertainty and improve accuracy of predictive toxicity assessments for PFAS.

Per- and polyfluoroalkyl substances (PFAS) are a class of over 8000 chemicals, many of which are persistent, bioaccumulative, and toxic to humans, livestock, and wildlife. Serum protein binding affinity is instrumental in understanding PFAS toxicity, yet experimental binding data is limited to only a few PFAS congeners. Previously, we demonstrated the usefulness of a high-throughput, in vitro differential scanning fluorimetry assay for determination of relative binding affinities of human serum albumin for 24 PFAS congeners from 6 chemical classes. In the current study, we used this assay to comparatively examine differences in human, bovine, porcine, and rat serum albumin binding of 8 structurally informative PFAS congeners from 5 chemical classes. With the exception of the fluorotelomer alcohol 1H, 1H, 2H, 2H-perfluorooctanol (6:2 FTOH), each PFAS congener bound by human serum albumin was also bound by bovine, porcine, and rat serum albumin. The critical role of the charged functional headgroup in albumin binding was supported by the inability of albumin of each species tested to bind 6:2 FTOH. Significant interspecies differences in serum albumin binding affinities were identified for each of the bound PFAS congeners. Relative to human albumin, perfluoroalkyl carboxylic and sulfonic acids were bound with greater affinity by porcine and rat serum albumin, and the perfluoroalkyl ether acid congener bound with lower affinity to porcine and bovine serum albumin. These comparative affinity data for PFAS binding by serum albumin from human, experimental model, and livestock species reduce critical interspecies uncertainty and improve accuracy of predictive bioaccumulation and toxicity assessments for PFAS.

IntroductionHematoporphyrin is a photosensitizer used in photodynamic therapy of various malignant diseases. It is carried to the cancer tissue by serum albumins. Spectrofluorimetric spectra of hematoporphyrin–serum albumin complexes were examined in vitro.Material and methodsThe chemicals were: hematoporphyrin, human serum albumin and bovine serum albumin. The spectra were recorded on a Kontron SFM-25 Instrument AG at two excitation wavelengths: ex = 280 nm and ex = 295 nm. The spectra of hematoporphyrin 1.5 × 10–5 M as well as spectra of complexes of hematoporphyrin–human serum albumin (1.5 × 10–5 M Hp – 1.25 × 10–6 M HSA) and hematoporphyrin–bovine serum albumin (1.5 × 10–5 M Hp – 3.5 × 10–7 M BSA) were recorded repetitively for 8 days and compared to the initial spectrum.ResultsFormation of a complex with human serum albumin extends the stability of the hematoporphyrin spectrum. This extension is greater at excitation ex = 295 nm. Different stability of complexes with bovine and human serum albumins most likely does not result from an actual lower stability of bovine serum albumin complexes, but from the fact that dissimilarity in the structure of both albumins enables additional spectroscopic observations within subdomain IB in the bovine serum albumin molecule.ConclusionsSpectrofluorimetric spectra are stable longer when hematoporphyrin forms a complex with human serum albumin. The present data may be important for understanding the mechanism of hematoporphyrin transportation to the target cancer tissue and effectiveness of photodynamic therapy.

Serum albumin first appeared in early vertebrates and is present in the plasma of all mammals. Its canonical structure supported by a conserved set of disulfide bridges is maintained in all mammalian serum albumins and any changes in sequence are highly correlated with evolution of the species. Previous structural investigations of mammalian serum albumins have only concentrated on human serum albumin (HSA), most likely as a consequence of crystallization and diffraction difficulties. Here, the crystal structures of serum albumins isolated from bovine, equine and leporine blood plasma are reported. The structure of bovine serum albumin (BSA) was determined at 2.47 Å resolution, two crystal structures of equine serum albumin (ESA) were determined at resolutions of 2.32 and 2.04 Å, and that of leporine serum albumin (LSA) was determined at 2.27 Å resolution. These structures were compared in detail with the structure of HSA. The ligand-binding pockets in BSA, ESA and LSA revealed different amino-acid compositions and conformations in comparison to HSA in some cases; however, much more significant differences were observed on the surface of the molecules. BSA, which is one of the most extensively utilized proteins in laboratory practice and is used as an HSA substitute in many experiments, exhibits only 75.8% identity compared with HSA. The higher resolution crystal structure of ESA highlights the binding properties of this protein because it includes several bound compounds from the crystallization solution that provide additional structural information about potential ligand-binding pockets.

Immune recognition of serum albumin. 11. Mouse antibodies against bovine serum albumin recognize the same antigenic sites that are recognized by rabbit antibodies

The interaction of the immobilized triazine dye Cibacron Blue 3G-A with rat, rabbit, sheep, goat, bovine and human serum albumins was studied by affinity gel electrophoresis. Dissociation constants were estimated in each instance and showed human serum albumin to have a significantly higher affinity for the dye than did albumin from any other species. Pretreatment of the defatted proteins with bilirubin (3 mol of bilirubin/mol of protein) did not increase the dissociation constants of the serum albumins, whereas pretreatment with palmitate (7 mol of palmitate/mol of protein) increased the dissociation constant in all cases: 3-fold for human serum albumin, 15-fold for other serum albumins. Increasing the bilirubin/albumin ratio (to 7:1) did not affect the dissociation constant of the albumins studied. Decreasing the palmitate/albumin ratio decreased the dissociation constant for human serum albumin, but did not affect those of bovine and rat albumins. Altering the chain length of the presaturating fatty acid dramatically changed the dissociation constant of both human and bovine serum albumins. Butyrate, hexanoate, octanoate and decanoate did not significantly influence the dissociation constants of bovine and human serum albumins for Cibacron Blue, whereas laurate, myristate and palmitate greatly increased the dissociation constant. These data are discussed in relationship to the behaviour of albumins during dye--agarose column chromatography. In Addendum the effect of nucleotide presaturation on the interaction between Bacillus stearothermophilus 6-phosphogluconate dehydrogenase and the immobilized triazine dyes Cibacron Blue 3G-A and Procion Red HE-3B was examined, and the implications for dye--ligand chromatography are discussed.

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 of 14C-riboflavin and then cleaved by CNBr, two peptides were obtained, containing the Trp-134 and Trp-212 residues, respectively. The incorporation of 14C-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.

The progressive escalation in the applications of bile salts in diverse fields has triggered research on their interaction with various biological macromolecules, especially with proteins. A proper understanding of the interaction process of bile salts, particularly in the lower concentrations range, with the serum albumin seems important since the normal serum concentration of bile salts is approximately in the micromolar range. The current study deals with a comprehensive and comparative analysis of the interaction of submicellar concentrations of sodium deoxycholate (NaDC) with two homologous transport proteins: bovine serum albumin (BSA) and human serum albumin (HSA). HSA and BSA with one and two tryptophans, respectively, provide the opportunity for an interesting comparison of tryptophan fluorescence behavior on interaction with NaDC. The study suggests a sequential interaction of NaDC in three discrete stages with the two proteins. A detailed study using warfarin and ibuprofen as site markers provides information about the sites of interaction, which is further confirmed by inclusive molecular dynamics simulation analysis. Moreover, the comparison of the thermodynamics and stability of the NaDC-serum albumin complexes confirms the stronger interaction of NaDC with BSA as compared to that with HSA. The differential interaction between the bile salt and the two serum albumins is further established from the difference in the extent of decrease in the esterase-like activity assay of the proteins in the presence of NaDC. Therefore, the present study provides important insight into the effect of submicellar concentrations of NaDC on the structure, stability, and activity of the two homologous serum albumins and thus can contribute not only to the general understanding of the complex nature of serum albumin-bile salt interactions but also to the design of more effective pharmaceutical formulations in the field of drug delivery and biomedical research.

Binding of parathion and paraoxon to bovine serum albumin (BSA) and human serum albumin (HSA) was studied by using equilibrium dialysis. The concentration of unbound organophosphate was determined from its anticholinesterase activity. Binding of parathion to BSA was shown to be reversible. The organophosphates interact with only one type of binding sites in BSA and HSA. The affinity constants at pH 7.2 and 4 degrees C for the interaction of BSA or HSA and parathion were found to be 2.7 X 10(6) and 1.5 X 10(6) M-1, respectively. The affinity constants for the interaction of the serum albumins and paraoxon were considerably lower, 6.0 X 10(3) and 1.6 X 10(4) M-1, respectively. Lowering the pH from 7.2 to 4.8 did not significantly affect the binding parameters. The great difference of affinity of the serum albumins to parathion and paraoxon is discussed with respect to the fate of parathion in the body.

The storage modulus, G', and aggregation of β-lactoglobulin AB and bovine serum albumin (BSA) were measured during 60 min of heating at 70 or 75 °C in a buffer simulating the whey protein concentrate environment. BSA formed stiffer gels than β-lactoglobulin and also aggregated (shown by loss of native protein by PAGE) and polymerized (shown by loss of residual monomeric protein by SDS−PAGE) more rapidly than β-lactoglobulin. Both BSA and β-lactoglobulin formed heat-induced hydrophobically bonded aggregates which may have been intermediates in the transformation of the native proteins into the disulfide-bonded gel networks. The gelation temperature (the temperature at which an appreciable increase in G' occurred during heating at 1 °C/min) of mixtures of BSA and β-lactoglobulin at a total protein concentration of 10% (w/v) increased from ∼72 °C, for BSA alone, to ∼84 °C, for β-lactoglobulin alone. When protein solutions were heated, BSA solutions formed stiffer gels at lower concentrations and at lower temperatures. When 10% mixtures of the two proteins were heated at 75 °C, the gelling behavior of the mixture was more akin to that of BSA when BSA was the major constituent in the mixture and vice versa. The results are consistent with the formation of intertwined and commingled homopolymers of BSA and β-lactoglobulin together with some heteropolymers. The proportion of homo- and heteropolymers in each mixture is probably dependent on the heating temperature and the composition of the mixture. Keywords: Whey protein concentrate; hydrophobic aggregation; disulfide bonding

Quantitation of species differences in albumin–ligand interactions for bovine, human and rat serum albumins using fluorescence spectroscopy: A test case with some Sudlow's site I ligands