Serum Albumin Complex Research Articles

Redox-mediated mechanisms and biological responses of copper-catalyzed reduction of the nitrite ion in vitro

During ischemia nitrite may be converted into nitric oxide (NO) by reaction with heme-carrying proteins or thiol-containing enzymes. NO acts as a regulator of vasodilation and protector against oxidative stress-induced tissue injuries. As a result of ischemia-induced oxidative stress, hypoxia and/or acidosis bivalent copper ions (Cu2+) can dissociate from their physiological carrier proteins. Reduced by the body’s own antioxidants, the resultant Cu1+ might represent an effective reductant of nitrite. Here we have evaluated in vitro copper-dissociation from copper/BSA (bovine serum albumin) complexes under ischemic conditions. Furthermore, using physiological concentrations, we have characterized the capacity of antioxidants and bivalent copper ions to serve as Cu1+-agitated catalytic sites for nitrite reduction and also the biological responses of this mechanism in vitro. We found that as a consequence of an acidic milieu and/or oxidative stress the copper-binding capacity of serum albumin strongly declined, leading to significant dissociation of copper ions into the ambient solution. At physiologically relevant pH-values Cu2+ ions in combination with physiologically available copper reductants (i.e., ascorbate, glutathione, Fe2+) significantly enhanced nitrite reduction and subsequent non-enzymatic NO generation under hypoxic but also normoxic conditions. Our data demonstrate for the first time that upon ischemic conditions carrier protein-dissociated copper ions combined with appropriate reductants may serve as Cu1+-driven catalytic sites for nitrite reduction, leading to the formation of biologically relevant NO formation. Thus, in addition to the action of heme proteins, copper-catalyzed non-enzymatic NO formation from nitrite might represent a further physiologically relevant vasodilating and NO-dependent protective principle to ischemic stress.

Characterization and antioxidant activity of bovine serum albumin and sulforaphane complex in different solvent systems

Modes and influencing factors of bovine serum albumin (BSA) and sulforaphane (SFN) interaction will help us understand the interaction mechanisms and functional changes of bioactive small molecule and biomacromolecule. This study investigated interaction mechanisms of BSA and SFN and associated antioxidant activity in three solvent systems of deionized water (dH2O), dimethyl sulfoxide (DMSO) and ethanol (EtOH), using Fourier transform infrared spectroscopy (FT-IR), fluorescence spectroscopy, synchronous fluorescence spectroscopy, DPPH and ABTS radical scavenging assays. The results revealed that SFN had ability to quench BSA's fluorescence in static modes, and to interact with BSA at both tyrosine (Tyr) and tryptophan (Trp) residues, while the Trp residues were highly sensitive, which was demonstrated by fluorescence at 340nm. Hydrophobic forces, hydrogen bonds and van der Waals interactions were all involved in BSA and SFN interaction, which were not significantly changed by three solvents. The binding constant values and binding site numbers were in a descending order of dH2O>DMSO>EtOH. The values of free energy change were in a descending order of dH2O>DMSO>EtOH, which indicated that the binding forces were in a descending order of dH2O>DMSO>EtOH. There was no significant difference in antioxidant activity between SFN and BSA–SFN. Moreover, three solvents had not significant influence on antioxidant activity of SFN and BSA–SFN.

Diffracted X-ray tracking for monitoring intramolecular motion in individual protein molecules using broad band X-ray

Diffracted X-ray tracking (DXT) enables the tilting and twisting motions of single protein molecules to be monitored with micro- to milliradian resolution using a highly brilliant X-ray source with a wide energy bandwidth. We have developed a technique to monitor single molecules using gold nanocrystals attached to individual protein molecules using the BL28B2 beamline at SPring-8. In this paper we present the installation of a single toroidal X-ray mirror at BL28B2 to focus X-rays in an energy range of 10-20 keV (ΔE/E = 82% for an X-ray with a wide energy bandwidth). With this beamline we tracked diffraction spots from gold nanocrystals over a wide angle range than that using quasi-monochromatic X-rays. Application of the wide angle DXT technique to biological systems enabled us to observe the on-site motions of single protein molecules that have been functionalized in vivo. We further extend the capability of DXT by observing the fractional tilting and twisting motions of inner proteins under various conditions. As a proof of this methodology and to determine instrumental performance the intramolecular motions of a human serum albumin complex with 2-anthracenecarboxylic acid was investigated using the BL28B2 beamline. The random tilting and twisting intramolecular motions are shown to be directly linked to the movement of individual protein molecules in the buffer solution.

Suppression of SERPINA1-albumin complex formation by galectin-3 overexpression leads to paracrine growth promotion of chronic myelogenous leukemia cells

Galectin-3 is induced in chronic myelogenous leukemia (CML) cells by co-culture with bone marrow stromal cells, making paracrine growth promotion of CML cells in conditioned medium (CM) from galectin-3 overexpressing CML cells more potent. We used gel filtration chromatography to demonstrate that the bovine SERPINA1-fetal bovine serum albumin (BSA) complex was specifically suppressed in CM from galectin-3 overexpressing cells. The SERPINA1-BSA complex as well as human plasma SERPINA1 inhibited the growth of CML cells, while exogenous galectin-3 partly offset this effect. These findings suggest that galectin-3 overexpression promotes paracrine growth of CML cells by interfering with the action of the growth inhibitory SERPINA1-albumin complex.

A comparative study: In vitro effects of EPA and DHA on immune functions of head-kidney macrophages isolated from large yellow croaker (Larmichthys crocea)

Comparative effects of different concentrations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on immune responses of head-kidney macrophages isolated from large yellow croaker were studied in vitro. After exposing to serum-free medium for 1 day, cultured cells were incubated in medium supplemented with graded levels of EPA or DHA (0, 5, 25, 100, 200 and 1000 μM, respectively) in the form of fatty acid bovine serum albumin (FA-BSA) complex for 12 h, 24 h and 36 h, respectively. Control samples were incubated in the absence of EPA or DHA (2% bovine serum albumin, BSA). Following stimulation, cell viability, lipid peroxidation, secretary phopholipase A2 (sPLA2) and prostaglandin E2 (PGE2) production as well as some immune parameters including phagocytosis, respiratory burst activity and interleukin 1β (IL-1β) production were determined. Results showed that EPA and DHA affected cell viability in dose-dependent and time-dependent manners. In particular, cell viability was significantly decreased after 24 h and 36 h incubation with 1000 μM EPA or DHA (P < 0.05). Higher levels of EPA (200 and 1000 μM) caused a significant increase in the production of malondialdehyde (MDA) (P < 0.05), while DHA did not significantly affect the MDA production. EPA significantly increased the intracellular superoxide anion synthesis which, on the contrary, was significantly reduced by DHA. Phagocytosis percentage (PP) values were significantly higher in treatments with 5 μM DHA (P < 0.05), but significantly decreased by 200 and 1000 μM EPA and DHA compared to the control group (P < 0.05). Decreased PGE2 production was produced by cells treated with relatively low doses of EPA or DHA. When high levels of stimulants (1000 μM EPA or DHA) were used, PGE2 levels were elevated and reached a significant level (P < 0.05). Both EPA and DHA significantly inhibited the production of sPLA2, where DHA exerted the more potent inhibitory effects than EPA. No pronounced effect was observed on IL-1β production among all the treatments, and IL-1β level in cell culture supernatant was fairly low (only approximately 6 pg/ml). Those findings suggested that EPA and DHA could influence the immunity and physiological conditions of macrophages from head kidney of large yellow croaker in vitro.

Spectroscopic Investigation on Sonocatalytic Damage of BSA Molecules by FeIII Complexes with Binary Organic Acid Ligands Under Ultrasonic Irradiation

The interaction between bovine serum albumin (BSA) and FeIII complexes with three binary organic acid (biorga) ligands, [FeIII(oxa)(H2O)4]+ (oxa = oxalic acid), [FeIII(pra)(H2O)4]+ (pra = propanedioic acid) and [FeIII(sua)(H2O)4]+ (sua = succinic acid), as well as the sonocatalytic damage of BSA in the presence of these three FeIII–biorga complexes under ultrasonic irradiation, were studied by UV–vis and fluorescence spectra. The experimental results show that the fluorescence quenching process of BSA caused by three FeIII–biorga complexes are all static quenching and the corresponding quenching rate constants (K q), equilibrium constants (K A) and the binding site numbers (n) were calculated. The results reveal that, under ultrasonic irradiation, the BSA molecules were obviously damaged by these FeIII–biorga complexes. In addition, the effects of several factors on the damage of BSA molecules were examined. The experimental results demonstrate that the damage degree of BSA increased with an increase of ultrasonic irradiation time, FeIII–biorga complex concentration, and ionic strength. In comparison, [FeIII(pra)(H2O)4]+ exhibited higher sonocatalytic activity than [FeIII(oxa)(H2O)4]+ and [FeIII(sua)(H2O)4]+. Finally, the extent of generation of \( \cdot {\text{O}}_{2}^{ - } \)and ·OH during sonocatalytic processes was estimated. Perhaps, the results will be significant for promoting sonodynamic treatment (SDT) of tumors at the molecular level.

Experimental-Like Affinity Constants and Enantioselectivity Estimates from Flexible Docking

Experimental-like affinity constants and enantioselectivity estimates, not predicted so far computationally, were obtained using a novel flexible modeling/docking combined strategy. The S- and R-warfarin-human serum albumin (HSA, site I) complexes were used as an interaction model. The process for a verified estimation includes the following: (i) ionized open chain forming at physiological pH (a recent focus); (ii) conformational search (molecular mechanics and Monte Carlo methods); (iii) rigid protein-flexible ligand docking (GlideXP) generating low energy paired S- and R-poses; (iv) graphical comparison against the X-ray crystal structure (unsatisfactory verification step); (v) quantum polarized ligand docking (insufficient verification step); (vi) induced fit docking (one pose satisfying the verification criterion; selection step); (vii) converting docking scores to affinity and enantioselectivity estimates (log K(S) = 5.43, log K(R) = 5.34, ES = K(S)/K(R) = 1.23) and numerical comparison against equivalent literature data from bioanalytical techniques (validation step); (viii) intermolecular forces explaining ES (hydrogen bonding and π-π interactions).

Chiral recognition of metalaxyl enantiomers by human serum albumin: evidence from molecular modeling and photophysical approach

Metalaxyl is an acylamine fungicide, belonging to the most widely known member of the amide group. This task is aimed to scrutinize binding region and spatial structural change of principal vector human serum albumin (HSA) complex with (R)-/(S)-metalaxyl by exploiting molecular modeling, steady-state and time-resolved fluorescence, and circular dichroism (CD) approaches. According to molecular modeling, (R)-metalaxyl is situated within subdomains IIA and IIIA and the affinity of site I with (R)-metalaxyl is greater than site II, whereas (S)-metalaxyl is only located at subdomain IIA and the affinity of (S)-metalaxyl with site I is superior compared with that with (R)-metalaxyl. This coincides with the competitive ligand binding, guanidine hydrochloride-induced unfolding of protein, and hydrophobic 8-anilino-1-naphthalenesulfonic acid experiments; the acting forces between (R)-/(S)-metalaxyl and HSA are hydrophobic, π-π interactions, and hydrogen bonds, as derived from molecular modeling. Fluorescence emission manifested that the complex of (R)-/(S)-metalaxyl to HSA is the formation of adduct with an affinity of 10(4) M(-1), which corroborates the time-resolved fluorescence that the static type was operated. Furthermore, the changes of far-UV CD spectra evidence the polypeptide chain of HSA partially unfolded after conjugation with (R)-/(S)-metalaxyl. Through this work, we envisage that it can offer central clues on the biodistribution, absorption, and bioaccumulation of (R)-/(S)-metalaxyl.

The influence of fatty acids on theophylline binding to human serum albumin. Comparative fluorescence study

Theophylline, popular diuretic, is used to treat asthma and bronchospasm. In blood it forms complexes with albumin, which is also the main transporter of fatty acids. The aim of the present study was to describe the influence of fatty acids (FA) on binding of theophylline (Th) to human serum albumin (HSA) in the high affinity binding sites. Binding parameters have been obtained on the basis of the fluorescence analysis.The data obtained for the complex of Th and natural human serum albumin (nHSA) obtained from blood of obese patients qualified for surgical removal of stomach was compared with our previous studies on the influence of FA on the complex of Th and commercially available defatted human serum albumin (dHSA).

In vitro effects of arachidonic acid on immune functions of head kidney macrophages isolated from large yellow croaker (Larmichthys crocea)

The experiments were conducted to determine the effects of arachidonic acid (ARA) on macrophages isolated from large yellow croaker head kidney. After exposing to serum-free medium for 1day, cultured cells were incubated in medium supplemented with graded levels of ARA (0, 5, 25, 100, 200 and 1000μM) in the form of fatty acid bovine serum albumin (FA-BSA) complex for 12h, 24h and 36h, respectively. Cells treated with 0μM ARA (2% bovine serum albumin, BSA) were included as control at each incubation time. Following stimulation, cell viability, lipid peroxidation, secretory phopholipase A2 (sPLA2) production and some immune parameters including phagocytosis, respiratory burst activity, prostaglandin E2 (PGE2) and Interleukin 1β (IL-1β) production were examined. Results showed that cell viability decreased with prolonged exposure and increasing ARA concentration. Especially after 36h, cell viability was significantly decreased by incubation with higher levels of ARA (200 and 1000μM) (P<0.01). In the meanwhile, higher levels of ARA (200 and 1000μM) caused a significant increase in the production of malondialdehyde (MDA) (P<0.05, P<0.01). Phagocytosis percentage (PP) values were significantly higher in treatments with 25 and 100μM ARA (P<0.05), but significantly lower in treatments with 1000μM ARA compared to the control group (P<0.01). Enhanced intracellular superoxide anion (O2−) synthesis was found in cells treated with each level of ARA with an exception of 1000μM ARA (P>0.05). The significantly promoted production of sPLA2 seemed to be irrelevant to increased ARA concentration, while PGE2 production was significantly increased in an ARA dose dependent way (P<0.01). No pronounced effect on IL-1β production was observed between the control and ARA group, and levels of IL-1β in cell culture supernatant were fairly low (only approximately 6pg/ml). These findings suggested that ARA could influence the immunity and physiological conditions of macrophages from head kidney of large yellow croaker in vitro.

The lipopolysaccharide-induced pro-inflammatory response in RAW264.7 cells is attenuated by an unsaturated fatty acid–bovine serum albumin complex and enhanced by a saturated fatty acid–bovine serum albumin complex

A 1:1 ratio of fatty acid (FA)-albumin complex was chosen to mimic physiological conditions, and the effects of FA-bovine serum albumin (BSA) complexes were tested in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Nitric oxide (NO) and various proteins/factors in RAW264.7 cells were quantified as follows: NO by the Griess assay; prostaglandin (PG) E(2), interleukin (IL)-6 and tumor necrosis factor (TNF)-α by ELISA; inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 by Western blotting; and NF-κB and CD14/TLR4 by Western blotting or flow cytometry. BSA- or FA-BSA-treated RAW264.7 cells without LPS stimulation did not show any significant changes in NO or the tested proteins/factors and thus did not have any pro-inflammatory responses. Pre-treatment with unsaturated FA-BSA complexes significantly decreased the production of LPS-induced NO, PGE(2), IL-6 and TNF-α, the expression of iNOS, COX-2 and CD14, IκB degradation and NF-κB translocation. On the contrary, pre-treatment with saturated FA-BSA complexes enhanced these LPS-induced pro-inflammatory factors and the subsequent responses. We concluded that unsaturated FA-BSA complexes, but not saturated FA-BSA complexes, exert an inhibitory effect on the LPS-induced pro-inflammatory response and that this effect may be partially mediated through suppression of the NF-κB signaling pathway. We suggest that an increase of unsaturated FA-BSA complexes may enhance the host's defense against bacterial infection.

G-protein coupled receptor-associated sorting protein 1 (GASP-1), a potential biomarker in breast cancer

An innovative “2-D high performance liquid electrophoresis” (2-D HPLE) technology was used to identify serum biomarkers associated with the early stage of breast cancer in addition to other more advanced stages. 2-D HPLE is a newly developed electrophoretic technology that separates 100s of serum albumin complexes on a polyvinyl membrane based on their surface charges. Association of cancer proteins or their fragments (biomarkers) with pre-existing albumin complexes in the blood of cancer patients results in altered mobility on the membrane. Using 2-D HPLE we identified that a specific fragment of G-protein coupled receptor-associated sorting protein 1 (GASP-1) was present in the sera of patients with early stage disease but absent in sera of normal patients. GASP-1 has been shown to modulate lysosomal sorting and functional down-regulation of a variety of G-protein coupled receptors in neuronal cells. However, no reports have linked GASP-1 to breast cancer pathogenesis. GASP-1 was detected in tumor extracts of 7 cases of Stage 2 and Stage 3 breast cancers, but not in adjacent normal tissue as revealed by western blot analysis using an antibody developed against a GASP-1 peptide identified by our 2-D HPLE technology. Using this antibody, we immunohistochemically detected over-expression of GASP-1 in all of 107 cases of archived ductal breast carcinoma tumor samples, while normal adjacent breast tissue from 12 cases of ductal carcinoma showed little or no staining. Additionally, all 10 cases of metastatic breast carcinoma present in lymph nodes were positive. Strong positive GASP-1 staining was observed in all tumor tissue including ductal carcinoma in situ (DCIS) and invasive ductal carcinoma. Additionally, we observed a wide spectrum of enhanced staining of premalignant ductal epithelial cells present in benign ducts and those found in atypical ductal hyperplasia (ADH). These studies identify GASP-1 as a potential new serum and tumor biomarker for breast cancer and suggest that GASP-1 may be a novel target for the development of breast cancer therapeutics.

Abstract 3183: The role of G-protein coupled receptor-associated sorting protein 1 (GASP-1) in breast cancer progression

Abstract An innovative “2-D High Performance Liquid Electrophoresis” (2-D HPLE) technology was used to identify serum biomarkers associated with the earliest stage of breast cancer in addition to other more advanced stages. 2-D HPLE is a newly developed electrophoretic technology that separates 100's of serum albumin complexes on a polyvinyl membrane based on their surface charges. Association of cancer proteins (biomarkers) with pre-existing albumin complexes in the blood of cancer patients results in altered mobility on the membrane. Using 2-D HPLE we identified that G-protein coupled receptor-associated sorting protein 1(GASP-1) was present in the sera of patients with early stage disease but absent in sera of normal patients. GASP-1 has been shown to modulate lysosomal sorting and functional down-regulation of a variety of G-protein coupled receptors in neuronal cells. However, no reports have linked GASP-1 to breast cancer pathogenesis. GASP-1 was detected in tumor extracts of 7 cases of stage 2 and stage 3 breast cancer but not in adjacent normal tissue as revealed by Western Blot analysis using an antibody developed against a GASP-1 peptide identified by our 2-D HPLE technology. Using this antibody, we immunohistochemically detected over-expression of GASP-1 in all 59 cases of archived ductal breast carcinoma tumor samples. When GASP-1 was silenced in MB-231 breast carcinoma, the cells grew more than 5 fold slower than corresponding vector controls. These studies identify GASP-1 as a potential new serum and tumor biomarker for breast cancer and suggest that GASP-1 may be a novel target for development of breast cancer therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3183. doi:10.1158/1538-7445.AM2011-3183

Characterization of the baicalein–bovine serum albumin complex without or with Cu 2+or Fe 3+ by spectroscopic approaches

The binding of baicalein to bovine serum albumin (BSA) in the absence and presence of Cu 2+ or Fe 3+ in aqueous solution has been studied by fluorescence, synchronous fluorescence, ultraviolet–visible (UV–vis) spectra, circular dichroism (CD) and the three-dimensional (3D) fluorescence at pH 7.40. The decrease of the binding constant in the presence of Cu 2+ or Fe 3+ may result from the competition of the metal ions and baicalein binding to BSA. The effect of baicalein on the conformation of BSA was analyzed using UV, CD, fluorescence and three-dimensional (3D) fluorescence. These results indicate that the binding of baicalein to BSA causes apparent change in the secondary structure of BSA, but does not affect the polarity around the chromophore molecule.

Interactions of Two Ethidium Derivatives with Calf Thymus DNA and Serum Albumins Using Fluorescence Quenching Method

In the present study, we reported the syntheses and investigation of the modes of binding to DNA and serum albumins of the two ethidium derivatives containing ethyl and methyl chloroformate of both amines at 3-and 8- positions. The interactions of calf thymus DNA (ct-DNA) and serum albumins (BSA and HSA) with the two derivatives, E4 and E5, were investigated using fluorescence quenching method and UV-visible absorption spectra. The obtained data suggest that used derivatives can interact strongly with DNA and serum albumins; there are the formation of derivative-DNA and serum albumin complexes and hydrophobic interactions as the predominant intermolecular forces in stabilizing for each complex. The Stern-Volmer quenching constants, binding constants, binding sites and the corresponding thermodynamic parameters ΔH, ΔS and ΔG were calculated at different temperatures. The results of absorption, ionic strength and iodide ion quenching suggested that the interaction mode of E4 and E5 with DNA was groove binding. Keywords: Fluorescence Quenching, Stern-Volmer, Calf thymus DNA, 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.

Chitosan–bovine serum albumin complex formation: A model to design an enzyme isolation method by polyelectrolyte precipitation

Interactions between a model protein (bovine serum albumin—BSA) and the cationic polyelectrolyte, chitosan (Chi), have been characterized by turbidimetry, circular dichroism and fluorescence spectroscopy. It has been found that the conformation of the BSA does not change significantly during the chain interaction between BSA and chitosan forming the non-covalently linked complex. The effects of pH, ionic strength and anions which modify the water structure around BSA were evaluated in the chitosan–BSA complex formation. A net coulombic interaction force between BSA and Chi was found as the insoluble complex formation decreased after the addition of NaCl. Around 80% of the BSA in solution precipitates with the Chi addition. A concentration of 0.05% (w/v) Chi was necessary to precipitate the protein, with a stoichiometry of 6.9 g BSA/g Chi. No modification of the tertiary and secondary structure of BSA was observed when the precipitate was dissolved by changing the pH of the medium. Chitosan proved to be a useful framework to isolate proteins with a slightly acid isoelectrical pH by means of precipitation.

Site-Selective Interactions: Squaraine Dye−Serum Albumin Complexes with Enhanced Fluorescence and Triplet Yields

We report the effect of steric factors of a few squaraine dyes, bis(2,4,6-trihydroxyphenyl)squaraine (1), bis(3,5-dibromo-2,4,6-trihydroxyphenyl)squaraine (2), and bis(3,5-diiodo-2,4,6-trihydroxyphenyl) squaraine (3), on their binding with human (HSA) and bovine (BSA) serum albumins employing photophysical, chiroptical, biophysical, and microscopic techniques. These dyes interact with serum albumins very efficiently and exhibit site selectivity, involving synergistic effects of hydrophobic, hydrogen bonding, and electrostatic interactions. The association constants of these complexes have been determined and are found to be 4.9 x 10(6) and 4.1 x 10(5) M(-1), respectively, for the dyes 2 and 3 with BSA, while HSA showed relatively higher association constants of 6.0 x 10(6) and 9.9 x 10(5) M(-1). Highly clear distinction in site-selective binding can be ascertained from time-resolved fluorescence, displacement cum fluorimetry, and circular dichroism (CD) studies. The increased affinity toward the major binding site (site II, domain III) over the relatively smaller binding site (site I, domain II) in the serum albumin with the increasing size of the heavy atoms present in 2 and 3 as compared to 1 indicates the importance of steric factors thereby confirming that the dye structure has a predominant role in deciding site selectivity. The distance between the energy donor and acceptor was calculated using Forster theory, which agrees well with the reported site 1 binding agent dansylamine. In contrast, no energy transfer was observed between tryptophan (Trp-214) present in domain II of the albumins and the dyes 2 and 3, indicating that these derivatives bind less efficiently at site I due to steric conatraints but preferentially bind at site II. Laser flash photolysis studies of the dyes 2 and 3 in the presence of HSA exhibited ca. 2.5-fold enhancements in the triplet lifetimes and quantum yields when compared to that obtained in buffer. The uniqueness of these dyes is that they show substituent size-dependent selectivity at site II of serum albumins and signal the event through "turn on" fluorescence intensity as well as enhanced triplet excited state lifetimes and quantum yields, thereby indicating their potential use as NIR noncovalent protein labeling and photodynamic therapeutic agents.

17Beta-Estradiol Enhances Leptin Expression in Human Placental Cells Through Genomic and Nongenomic Actions1

The process of embryo implantation and trophoblast invasion is considered the most limiting factor in the establishment of pregnancy. Leptin was originally described as an adipocyte-derived signaling molecule for the central control of metabolism. However, it has been suggested that leptin is involved in other functions during pregnancy, particularly in the placenta, where it was found to be expressed. In the present work, we have found a stimulatory effect of 17beta-estradiol (E(2)) on endogenous leptin expression, as analyzed by Western blot, in both the BeWo choriocarcinoma cell line and normal placental explants. This effect was time and dose dependent. Maximal effect was achieved at 10 nM in BeWo cells and 1 nM in placental explants. The E(2) effects involved the estrogen receptor, as the antagonist ICI 182 780 inhibited E(2)-induced leptin expression. Moreover, E(2) treatment enhanced leptin promoter activity up to 4-fold, as evaluated by transient transfection with a plasmid construction containing the leptin promoter region and the reporter gene luciferase. This effect was dose dependent. Deletion analysis demonstrated that a minimal promoter region between -1951 and -1847 bp is both necessary and sufficient to achieve E(2) effects. Estradiol action involved estrogen receptor 1, previously known as estrogen receptor alpha, as cotransfection with a vector encoding estrogen receptor 1 potentiated the effects of E(2) on leptin expression. Moreover, E(2) action probably involves membrane receptors too, as treatment with an estradiol-bovine serum albumin complex partially enhanced leptin expression. The effects of E(2) could be blocked by pharmacologic inhibition of MAPK and the phosphoinositide-3-kinase (PI3K) pathways with 50 microM PD98059 and 0.1 microM Wortmannin, respectively. Moreover, cotransfection of dominant negative mutants of MAP2K or MAPK blocked E(2) induction of leptin promoter. On the other hand, E(2) treatment promoted MAPK1/MAPK3 and AKT phosphorylation in placental cells. In conclusion, we provide evidence suggesting that E(2) induces leptin expression in trophoblastic cells, probably through genomic and nongenomic actions via crosstalk between estrogen receptor 1 and MAPK and PI3K signal transduction pathways.

Interaction of coomassie brilliant blue G250 with human serum albumin: Probing of the binding mechanism and binding site by spectroscopic and molecular modeling methods

Abstract The interaction between coomassie brilliant blue G250 and human serum albumin was investigated by spectroscopic methods such as fluorescence quenching, synchronous fluorescence , 3D fluorescence spectra, circular dichroism spectra and UV–vis absorption as well as molecular modeling. The fluorescence quenching of human serum albumin by coomassie brilliant blue G250 was attributed to static interaction. The binding reaction was mainly enthalpy-driven. Both van der Waals and hydrogen bonding forces played major roles in stabilizing the coomassie brilliant blue G250–human serum albumin complex. The Stern–Volmer quenching constant ( K SV ) and corresponding thermodynamic parameters (Δ H Θ , Δ G Θ and Δ S Θ ) were determined. Site marker competitive experiments indicated that coomassie brilliant blue G250 bound to site I (subdomain IIA) of human serum albumin. Molecular docking study further confirmed the binding mode obtained by experimental study. The conformational investigation demonstrated very minor micro-environmental and conformational changes in human serum albumin molecules.