Homologous Serum Albumin Research Articles

Insight into the Effect of Submicellar Concentrations of Sodium Deoxycholate on the Structure, Stability, and Activity of Bovine and Human Serum Albumin: An Interesting Comparison between Single and Double Tryptophan Proteins.

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.

Interactions between hydroxylated polycyclic aromatic hydrocarbons and serum albumins: multispectral and molecular docking analyses.

Hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) can bind to serum albumin and influence their distribution and elimination in organisms. Here, multispectral analysis and molecular docking methods were used to investigate the binding mechanism of two OH-PAHs, 1-hydroxyphenanthrene (1-OHPhe) and 9-hydroxyphenanthrene (9-OHPhe), with two homologous serum albumins, human serum albumin (HSA) and bovine serum albumin (BSA). The quenching constants of HSA with 1-OHPhe and 9-OHPhe were much larger than those for BSA. Energy transfer from the tryptophan (Trp) residues in HSA to 1-OHPhe and 9-OHPhe was more probable than from Trp in BSA. The interactions of 1-OHPhe and 9-OHPhe with Trp in HSA and BSA altered the microenvironment of Trp. Molecular docking results revealed that the binding modes and binding forces of 1-OHPhe and 9-OHPhe with HSA and BSA were different. The two OH-PAHs were used as fluorescent probes to analyze the microenvironmental hydrophobicities of HSA and BSA, which were distinctly different. The structural difference between HSA and BSA induced significant variations in their binding behaviour with 1-OHPhe and 9-OHPhe. Moreover, HSA was more susceptible to 1-OHPhe and 9-OHPhe than BSA. This work suggests that the differences between the two serum albumins should be considered in related studies.

Is the Sudlow site I of human serum albumin more generous to adopt prospective anti-cancer bioorganic compound than that of bovine: A combined spectroscopic and docking simulation approach

A comparative biophysical study on the individual conformational adaptation embraced by two homologous serum albumins (SA) (bovine and human) towards a potential anticancer bioorganic compound 2-(6-chlorobenzo[d] thiazol-2-yl)-1H-benzo[de] isoquinoline-1,3(2H)- dione (CBIQD) is apparent from the discrimination in binding behavior and the ensuing consequences accomplished by combined in vitro optical spectroscopy, in silico molecular docking and molecular dynamics (MD) simulation. The Sudlow site I of HSA although anion receptive, harbors neutral CBIQD in Sudlow site I (subdomain IIA, close to Trp) of HSA, while in BSA its prefers to snugly fit into Sudlow site II (subdomain IIIA, close to Tyr). Based on discernable diminution of HSA mean fluorescence lifetime as a function of biluminophore concentration, facile occurrence of fluorescence resonance energy transfer (FRET) is substantiated as the probable quenching mechanism accompanied by structural deformations in the protein ensemble. CBIQD establishes itself within HSA close to Trp214, and consequently reduces the micropolarity of the cybotactic environment that is predominantly constituted by hydrophobic amino acid residues. The stronger association of CBIQD with HSA encourages an allosteric modulation leading to slight deformation in its secondary structure whereas for BSA the association is comparatively weaker. Sudlow site I of HSA is capable to embrace a favorable conformation like malleable gold to provide room for incoming CBIQD, whereas for BSA it behaves more like rigid cast-iron which does not admit any change thus forcing CBIQD to occupy an altogether different binding location i.e. the Sudlow site II. The anticancer CBIQD is found to be stable within the HSA scaffold as vindicated by root mean square deviation (RMSD) and root mean square fluctuation (RMSF) obtained by MD simulation. A competitively inhibited esterase-like activity of HSA upon CBIQD binding to Lys199 and Arg257 residues, plausibly envisions that similar naphthalimide based prodrugs, bearing ester functionality, can be particularly activated by Sudlow site I of HSA. The consolidated spectroscopic research described herein may encourage design of naphthalimide based pro-drugs for effective in vivo biodistribution using HSA-based drug delivery systems.

Biophysical insight into the interaction mechanism of plant derived polyphenolic compound tannic acid with homologous mammalian serum albumins

Numerous phenolic compounds have been reported in the last decade that have a good antioxidant property and interaction affinity towards mammalian serum albumins. In the present study, we have utilized mammalian serum albumins as a model protein to examine their comparative interaction property with polyphenolic compound tannic acid (TA) by using various spectroscopic and calorimetric methods We have also monitored the esterase and antioxidant activity of mammalian serum albumins in the absence and presence of TA. The obtain results recommended that the TA have a good binding affinity (∼104 to 106M−1) towards mammalian serum albumins and shows double sequential binding sites, which depends on the concentration of TA that induced the conformational alteration which responsible for the thermal stability of proteins. Binding affinity, structural transition and thermodynamic parameters were calculated from spectroscopic and calorimetric method reveals that non-covalent interaction causes partial conformational alteration in the secondary structure of protein ie.; increase in α-helical content with decrease in β-sheet, random coil and other structure. Meanwhile, we have found that esterase activities of serum albumins were also stabilized against hydrolysis and shows higher antioxidant activity in the presence of TA because albumins its self have an immense antioxidant activity beside TA.

Chemical Probes and Engineered Constructs Reveal a Detailed Unfolding Mechanism for a Solvent-Free Multidomain Protein.

Despite the growing application of gas-phase measurements in structural biology and drug discovery, the factors that govern protein stabilities and structures in a solvent-free environment are still poorly understood. Here, we examine the solvent-free unfolding pathway for a group of homologous serum albumins. Utilizing a combination of chemical probes and noncovalent reconstructions, we draw new specific conclusions regarding the unfolding of albumins in the gas phase, as well as more general inferences regarding the sensitivity of collision induced unfolding to changes in protein primary and tertiary structure. Our findings suggest that the general unfolding pathway of low charge state albumin ions is largely unaffected by changes in primary structure; however, the stabilities of intermediates along these pathways vary widely as sequences diverge. Additionally, we find that human albumin follows a domain associated unfolding pathway, and we are able to assign each unfolded form observed in our gas-phase data set to the disruption of specific domains within the protein. The totality of our data informs the first detailed mechanism for multidomain protein unfolding in the gas phase, and highlights key similarities and differences from the known solution-phase pathway.

Concentration-dependent reversible self-oligomerization of serum albumins through intermolecular β-sheet formation.

Proteins inside a cell remain in highly crowded environments, and this often affects their structure and activity. However, most of the earlier studies involving serum albumins were performed under dilute conditions, which lack biological relevance. The effect of protein-protein interactions on the structure and properties of serum albumins at physiological conditions have not yet been explored. Here, we report for the first time the effect of protein-protein and protein-crowder interactions on the structure and stability of two homologous serum albumins, namely, human serum albumin (HSA) and bovine serum albumin (BSA), at physiological conditions by using spectroscopic techniques and scanning electron microscopy (SEM). Concentration-dependent self-oligomerization and subsequent structural alteration of serum albumins have been explored by means of fluorescence and circular dichroism spectroscopy at pH 7.4. The excitation wavelength (λex) dependence of the intrinsic fluorescence and the corresponding excitation spectra at each emission wavelength indicate the presence of various ground state oligomers of serum albumins in the concentration range 10-150 μM. Circular dichroism and thioflavin T binding assay revealed formation of intermolecular β-sheet rich interfaces at high protein concentration. Excellent correlations have been observed between β-sheet content of both the albumins and fluorescence enhancement of ThT with protein concentrations. SEM images at a concentration of 150 μM revealed large dispersed self-oligomeric states with sizes vary from 330 to 924 nm and 260 to 520 nm for BSA and HSA, respectively. The self-oligomerization of serum albumins is found to be a reversible process; upon dilution, these oligomers dissociate into a native monomeric state. It has also been observed that synthetic macromolecular crowder polyethylene glycol (PEG 200) stabilizes the self-associated state of both the albumins which is contrary to expectations that the macromolecular crowding favors compact native state of proteins.

Targeting proteins with toxic azo dyes: a microcalorimetric characterization of the interaction of the food colorant amaranth with serum proteins.

The interaction of amaranth with two homologous serum albumins from human and bovine (HSA and BSA) was studied by microcalorimetry. The binding stoichiometry for the complexation of amaranth to both BSA and HSA was around 1, and the equilibrium constants were (5.79 ± 0.07) × 10(5) and (1.76 ± 0.05) × 10(5) M(-1), respectively. The binding reaction to HSA at 298.15 K was driven by a large negative enthalpic contribution and a small but positive entropic contribution, while to BSA, it was entirely enthalpy-driven and the entropic contribution was unfavorable. Parsing of the standard molar Gibbs energy revealed that the complexation was dominated by non-polyelectrolytic forces. Temperature-dependent isothermal titration calorimetry studies revealed that the enthalpic contribution increased and the entropic contribution decreased with the rise in the temperature but the Gibbs energy change remained almost unaltered. Differential scanning calorimetry results revealed that the binding reaction stabilized the serum albumins significantly against thermal unfolding.

Crowding-Induced Quenching of Intrinsic Tryptophans of Serum Albumins: A Residue-Level Investigation of Different Conformations

Macromolecular crowding has been known to influence the global conformational landscape of proteins in various ways. However, only a few studies have focused their attention on the local perturbations that can occur in the presence of crowding agents. Here we have used the sensitivity of the tryptophan (Trp) fluorescence of two homologous serum albumins (BSA and HSA) to its (Trp) surroundings to monitor the local changes in the immediate proximity of the intrinsic fluorophore. Using the tool of fluorescence quenching we have shown that the commonly used synthetic macromolecular crowders (e.g., Dextran 6, Dextran 40, Ficoll 70, and PEG 8000) can bring about dramatic conformational modulations in the two proteins. Moreover, the nature of perturbation was observed to be largely dependent on the specific crowding agent used, with Dextran 6 showing the maximum effect while PEG 8000 showed the least. Additionally the extent of local structure modulation was found to be the largest either in the native state of ...

Spectroscopic Investigation of the Effect of Salt on Binding of Tartrazine with Two Homologous Serum Albumins: Quantification by Use of the Debye–Hückel Limiting Law and Observation of Enthalpy–Entropy Compensation

Formation of ion pair between charged molecule and protein can lead to interesting biochemical phenomena. We report the evolution of thermodynamics of the binding of tartrazine, a negatively charged azo colorant, and serum albumins with salt. The dye binds predominantly electrostatically in low buffer strengths; however, on increasing salt concentration, affinity decreases considerably. The calculated thermodynamic parameters in high salt indicate manifestation of nonelectrostatic interactions, namely, van der Waals force and hydrogen bonding. Site-marker competitive binding studies and docking simulations indicate that the dye binds with HSA in the warfarin site and with BSA at the interface of warfarin and ibuprofen binding sites. The docked poses indicate nearby amino acid positive side chains, which are possibly responsible for electrostatic interaction. Using the Debye-Hückel interionic attraction theory for binding equilibria, it is shown that, for electrostatic binding the calculated free energy change increases linearly with square root of ionic strength. Also UV-vis, fluorescence, CD data indicate a decrease of interaction with salt concentration. This study quantitatively relates how ionic strength modulates the strength of the protein-ligand electrostatic interaction. The binding enthalpy and entropy have been found to compensate one another. The enthalpy-entropy compensation (EEC), general property of weak intermolecular interactions, has been discussed.

Optical Spectroscopic Exploration of Binding of Cochineal Red A with Two Homologous Serum Albumins

Cochineal Red A is a negatively charged synthetic azo food colorant and a potential carcinogen. We present here the study of binding of Cochineal Red A with two homologous serum albumins, human (HSA) and bovine (BSA), in aqueous pH 7.4 buffer by optical spectroscopic techniques. Protein intrinsic fluorescence quenching by Cochineal Red A occurs through ground-state static interaction and its binding with BSA is stronger than with HSA. The magnitudes of thermodynamic parameters suggest that dye binding occurs principally via electrostatic complexation. Site-marker competitive binding shows that Cochineal Red A binds primarily to site I of serum albumins. Circular dichroic spectra indicate that dye binding results in some conformational modification of serum albumins. Increased ionic strength of the medium results in lowering of binding. This study provides an important insight into possible means of removal of dye toxicity.

Luminol Fluorescence Quenching in Biomimicking Environments: Sequestration of Fluorophore in Hydrophobic Domain

The photophysical behavior of luminol (LH(2)) was studied in a variety of biologically relevant systems ranging from surfactants, cyclodextrin, and proteins using steady-state and time-resolved fluorescence spectroscopy. It was shown that, out of two possible LH(2) conformers present in solution, the sequestration of relatively less polar structure into the hydrophobic domain of biological media is the primary reason for decrease in fluorescence intensity. The efficacy of LH(2) fluorescence quenching is substantially higher in micellar subdomain of cationic surfactant and depends on the nature of the headgroup. The thermodynamic parameters like enthalpy (ΔH) and entropy (ΔS) change, etc., corresponding to the binding of LH(2) in the model water-soluble protein, bovine serum albumin (BSA), were estimated by performing the fluorescence titration experiment at different temperatures. The involvement of subdomain IA and IIA of BSA in LH(2) binding was confirmed from the ligand replacement process with bilirubin (BIL). The difference in ligand binding with structurally homologous human serum albumin (HSA) is discussed in terms of positive cooperativity among these two binding domains of BSA with a Hill coefficient (n(H)) value of 2.26 ± 0.18 and a half-maximal concentration (K(0.5)) of 5.74 ± 0.23 μM at 298 K.

Secondary Structure of the Homologous Proteins, A A-Fetoprotein and Serum Albumin, from their Circular Dichroism and Infrared Spectra

Secondary structure composition of two homologous proteins, alpha-fetoprotein and serum albumin, has been compared using circular dichroism and Fourier-transform infrared spectroscopy. CD-only analysis gives an accurate a-helical content for SA, but underestimates the predicted value for AFP. In contrast, FTIR-only analysis underestimates the helical content of SA. However, analysis of hybrid IR-CD spectra gives more accurate values for secondary structure composition for both AFP and SA and shows that the secondary structures of these proteins are the same.

Kinetics of Human and Bovine Serum Albumin Adsorption at Silica–Titania Surfaces

The interaction of proteins with surfaces is important in separation and purification procedures as well as in metabolism and its regulation. The degree of binding to a given surface in principle depends on the precise amino acid composition of the protein, although very little is presently known about the relationship between amino acid sequence and binding. Here we report accurate measurements of the kinetics of adsorption of two closely homologous serum albumins (human and bovine) to a hydrated metal oxide surface, using an accurate integrated optics technique. Marked differences between the two proteins are observed. The results are analyzed using a model involving two bound forms, reversible and irreversible. The two forms are identified as two orientations of the protein with respect to the surface which make differing numbers of hydrogen bonds to the surface. These numbers were estimated on the basis of the measured desorption rate constants. The interfacial binding energy was calculated from the quotient of the adsorption and desorption rate constants and compared with the value calculated from surface energy available data. Remarkably, substitution of phosphate buffer for HEPES buffer causes dramatic changes in the adsorption, abolishing the irreversible mode completely for human serum albumin.

Pharmacokinetics of Succinylated Serum Albumin in Wistar Rats and Cynomolgus Monkeys: Implications for Dosage Regimens in the Therapy of HIV Infection.

Succinylation of serum albumin produces a negatively charged protein with a potent anti-HIV-1/2 and anti-SIVmac activity. The in vitro IC50 values of Suc-HSA against the respective primate lentiviruses are in the low nanomolar concentration range. Succinylated homologous serum albumin was given intravenously at four different doses, ranging from 0.1 to 10 mg/kg to Wistar rats and Cynomolgus monkeys. Plasma samples were assayed for their drug content using iodinated proteins. The pharmacokinetics parameters were calculated by a single compartment model, taking into account a potential saturable elimination process. In rats as well as in monkeys succinylated serum albumin clearly showed dose-dependent kinetics. The rate of Suc-SSA elimination from the bloodstream in macaques could be described by a Vmax of 11.7 ± 0.2 µg/min kg-(1) and a K. of 0.40 ± 0.06 µg/mL (5.5 nM). The elimination of Suc-RSA in rats was characterized by a 10-fold higher V. of 112 ± 29 µg/min kg(-1) and a much higher K. of 25 ± 9 µg/mL (340 nM). The volume of distribution was about the plasma volume for both species. In rats, no significant differences were found between the kinetic parameters of Suc-RSA, Suc-HSA, or Suc-SSA. Histochemical staining of tissue sections obtained from the liver, spleen, kidneys, and different lymph nodes showed that endothelial cells and macrophages from the liver and spleen are involved in the clearance of the negatively charged albumins. Since replication of HIV mainly takes place in the lymphoid tissue, uptake of succinylated albumin in this system may imply an interesting therapeutic aspect of the negatively charged albumins.

Induction and immunochemical properties of a novel anti-antibody

This paper describes results which characterize an induced antibody in normal outbred rabbits which we have, for convenience, called parareactant (PR). PR resulting from autoimmunization of rabbits with either keyhole limpet hemocyanin-anti-tetanus toxoid F(ab') 2 or with tetanus toxoid-anti-tetanus toxoid F(ab') 2 complexes was studied. PR activity was directed solely to autologous, homologous or heterologous F(ab') 2 fragments regardless of their specificity. PR failed to react with intact antibodies or with antigen-antibody complexes consisting of homologous antibody bound to specific antigen. Radioimmunoassay and ELISA inhibition assays showed that reactivity between PR and autologous anti-tetanus toxoid F(ab') 2 or homologous anti-bovine serum albumin F(ab') 2 fragments was specifically inhibited with antigen. Anti-allotypic antibodies specific for a2 and b6 markers strongly inhibited binding of 125I-anti-micrococcal carbohydrate F(ab') 2 (a2, b6) with PR (a3, b4, b5). PR specificity thus appears to be directed against non-idiotypic determinants present in Fv regions. Affinity immunoblotting was used to analyze clonality of PR in the sera collected from individual rabbits during the course of an active immune response. PR-positive sera displayed clonally restricted spectrotype patterns. PR molecules were predominantly IgG with isoelectric points of 5.9–6.8. These results strongly suggest that these PR molecules are coded by a small number of V region genes.

Sensitization induced in guinea pigs with beta-propiolactone-treated serum albumin: experimental evidence for the cause of allergic reactions in humans receiving human diploid cell rabies vaccines.

beta-Propiolactone-treated (BPL-T) homologous serum albumin caused anaphylaxis in guinea pigs with a frequency and severity equal to that of guinea pigs inoculated with human albumin. Untreated guinea pig serum albumin did not cause any reactions in these animals. Some recipients of current rabies vaccine produced in human diploid cells available in the USA develop systemic allergic reactions, usually following booster immunization. The BPL-T human albumin component of the vaccines was believed to be the cause of the complications. Our studies support this conclusion.

Immune modulation of connective tissue functions: Studies on the production of collagen synthesis inhibitory factor by populations of human peripheral blood mononuclear cells

It has been shown previously that a soluble factor(s) from human peripheral blood mononuclear cells was capable of specifically suppressing collagen synthesis by normal human dermal fibroblasts (S. A. Jimenez, W. McArthur and J. Rosenbloom, J. Exp. Med. 150, 1421, 1979). In this communication, the cell sources and the conditions for synthesis of this collagen synthesis inhibitory factor (CSIF) are identified. CSIF production by mononuclear cells was directly related to the number of cells in culture and was significantly enhanced by a variety of mitogens and by antigens. Homologous serum or bovine serum albumin was required for CSIF production and maximal levels were reached 48 hr after stimulation. Thymus-derived lymphocytes appeared to be the main cells responsible for CSIF synthesis but B lymphocytes also produced the factor in response to proper B-cell mitogens. Preparations of plastic-adherent mononuclear cells were also found to produce increased CSIF but it was not possible to exclude completely the presence of T lymphocytes in these preparations and therefore, the cell source of CSIF in these preparations was not clearly established. Through the use of metabolic inhibitors it was shown that CSIF production required de novo protein synthesis but not cell division. Indo-methacin had no effect on either the production of CSIF or on CSIF-mediated inhibition of collagen synthesis. The results indicate that CSIF has the classic characteristics of a lymphokine and suggest a mechanism by which the immune response could modulate connective tissue function.

The Renal Clearance of Albumin following Intravenous Loading of Homologous Albumin in Conscious Rats

Tubular reabsorption of albumin was studied in conscious rats by measuring the renal excretion at various plasma concentrations. The plasma albumin concentration was raised by loading the animals with homologous serum albumin. By using this procedure an average plasma concentration of 54 g/l could be reached, which is about twice the physiological plasma albumin concentration in untreated rats. Throughout the experiments the glomerular filtration rate was determined by the total plasma slope clearance of 51Cr-EDTA. Following the albumin load, the 51Cr-EDTA clearance remained at 54.8 +/- 12.2 ml/min/m2 body surface. By correlating renal albumin excretion and plasma albumin concentration, the sieving coefficient of albumin, phi, of 2.35 x 10(-4), the renal albumin threshold of 38 +/- 5 g/l, and the maximal tubular transport rate of albumin of 9.6 +/- 2.6 micrograms/min/g kidney weight could be calculated (mean +/- SD). There were some variations among the animals, resulting in a fairly large standard deviation. However, these values are in agreement with recent micropuncture data.

Changes in lung ultrastructure following heterologous and homologous serum albumin infusion in the treatment of hemorrhagic shock

Changes in lung ultrastructure following heterologous and homologous serum albumin infusion in the treatment of hemorrhagic shock

Changes in lung ultrastructure following heterologous and homologous serum albumin infusion in the treatment of hemorrhagic shock.

The object of this study was to compare the ultrastructure pulmonary effects of the infusion of homologous and heterologous serum albumin solution in the treatment of hemorrhagic shock in baboons. Adult baboons subjected to hemorrhagic shock were resuscitated with either baboon serum albumin, human serum albumin, or Ringer's lactate solution. The lungs were fixed in vivo with potassium pyroantimony, a solution which produces electron dense interstitial precipitation of sodium. The lungs from animals resuscitated with baboon serum albumin showed evidence of interstitial edema, including dispersion of collagen fibers, interstitial smudging and increased interstital sodium concentrations. Similar changes were seen following human serum albumin infusions. Lung tissue from animals treated with Ringer's lactate solution showed minimal changes from normal. These results suggest that interstitial pulmonary edema develops after either homologous or heterologous serum albumin infusion in the treatment of hemorrhagic shock in baboons.