The drug binding site of human α1-acid glycoprotein: Insight from induced circular dichroism and electronic absorption spectra

Human α₁-acid glycoprotein (AGP) is an important drug binding plasma protein which affects pharmacokinetical properties of various therapeutic agents. For the first time, interpretation of the induced circular dichroism (ICD) spectra of drug–AGP complexes is presented yielding valuable information on the protein binding environment. ICD spectra were obtained by novel ligands of which AGP induced optical activity have never been reported (primaquine, mefloquine, propranolol, terazosin, carbamazepine, rhodamine B) and by re-investigation of ICD spectra of protein-bound drugs published earlier (chlorpromazine, dipyridamole, prazosin). Spectroscopic features of the ICD and absorption bands of drugs combined with native AGP indicated chiral non-degenerate exciton coupling between the guest chromophore and the indole ring of an adjacent tryptophan (Trp) residue. Results of additional CD experiments performed by using recombinant AGP mutants showed no changes in the ligand binding ability of W122A in sharp contrast with the W25A which was unable to induce extrinsic CD signal with either ligand. Thus, these findings unequivocally prove that, likely via π–π stacking mechanism, Trp25 is essentially involved in the AGP binding of drugs studied here as well as of related compounds. Survey of the AGP binding data published in the literature support this conclusion. Our results provide a fast and efficient spectroscopic tool to determine the inclusion of ligand molecules into the β-barrel cavity of AGP where the conserved Trp25 is located and might be useful in ligand-binding studies of other lipocalin proteins.     

The drug binding site of human alpha1-acid glycoprotein: insight from induced circular dichroism and electronic absorption spectra

Human alpha(1)-acid glycoprotein (AGP) is an important drug binding plasma protein which affects pharmacokinetical properties of various therapeutic agents. For the first time, interpretation of the induced circular dichroism (ICD) spectra of drug-AGP complexes is presented yielding valuable information on the protein binding environment. ICD spectra were obtained by novel ligands of which AGP induced optical activity have never been reported (primaquine, mefloquine, propranolol, terazosin, carbamazepine, rhodamine B) and by re-investigation of ICD spectra of protein-bound drugs published earlier (chlorpromazine, dipyridamole, prazosin). Spectroscopic features of the ICD and absorption bands of drugs combined with native AGP indicated chiral non-degenerate exciton coupling between the guest chromophore and the indole ring of an adjacent tryptophan (Trp) residue. Results of additional CD experiments performed by using recombinant AGP mutants showed no changes in the ligand binding ability of W122A in sharp contrast with the W25A which was unable to induce extrinsic CD signal with either ligand. Thus, these findings unequivocally prove that, likely via pi-pi stacking mechanism, Trp25 is essentially involved in the AGP binding of drugs studied here as well as of related compounds. Survey of the AGP binding data published in the literature support this conclusion. Our results provide a fast and efficient spectroscopic tool to determine the inclusion of ligand molecules into the beta-barrel cavity of AGP where the conserved Trp25 is located and might be useful in ligand-binding studies of other lipocalin proteins.     

Multiple ligand-binding properties of the lipocalin member chicken α1-acid glycoprotein studied by circular dichroism and electronic absorption spectroscopy: The essential role of the conserved tryptophan residue

Multiple ligand-binding properties of the 30-kDa chicken α₁-acid glycoprotein (cAGP), a member of the lipocalin protein family, were investigated for the first time by using circular dichroism (CD) and UV/Vis absorption spectroscopy methods. By measuring induced CD (ICD) spectra, high-affinity binding (K_a ≈ 10⁵–10⁶ M⁻¹) of several drugs, dyes and natural compounds to cAGP was demonstrated including antimalarial agents (quinacrine, primaquine), phenotiazines (chlorpromazine, methylene blue), propranolol, non-steroidal antiinflammatory drugs (ketoprofen, diclofenac), tamoxifen, diazepam, tacrine, dicoumarol, cationic dyes (auramine O, thioflavine T, ethidium bromide), benzo[a]pyrene, l-thyroxine, bile pigments (bilirubin, biliverdin), alkaloids (piperine, aristolochic acid), saturated and unsaturated fatty acids. Analysis of the extrinsic CD spectra with the study of the covalently modified protein and CD displacement experiments revealed that a single Trp26 residue of cAGP conserved in the whole lipocalin family is part of the binding site, and it is essentially involved in the ligand-binding process via π–π stacking interaction resulting in the appearance of strong induced CD bands due to the non-degenerate intermolecular exciton coupling between the π–π* transitions of the stacked indole ring–ligand chromophore. The finding that cAGP is able to accommodate a broad spectrum of ligands belonging to different chemical classes suggests that its core β-barrel cavity is unusually wide containing overlapping sub-sites. Significance of these new data in understanding of the ligand-binding properties of other lipocalins, especially that of human AGP, and potential practical applications are briefly discussed. Overall, cAGP serves as a simple, ultimate model to extend our knowledge on ligand-binding properties of lipocalins and to study the role of tryptophan residues in molecular recognition processes.     

Analysis of binding interaction between the natural apocarotenoid bixin and human serum albumin by circular dichroism and fluorescence spectroscopy

Bixin is an important, pharmacologically active dietary cis-carotenoid, but its interaction with potential macromolecular targets is completely unexplored. This work was aimed to study the binding of bixin to human serum albumin (HSA), the most abundant protein in blood plasma. Circular dichroism (CD) spectroscopy in combination with UV/VIS absorption spectroscopy and fluorescence quenching techniques were applied. Appearance of induced CD bands in the UV- and VIS-absorption spectral regions indicated the formation of non-covalent carotenoid-albumin complexes. Shape and spectral position of the extrinsic Cotton effects suggested the binding of a single bixin molecule to HSA in chiral conformation. Scatchard and non-linear regression analyses of CD titration data resulted in similar values for the association constant (Ka = 6.6 and 4.6x10(5) M(-1), resp.) and for the number of binding sites (n = 1). The binding interaction was independently confirmed by fluorescence-quenching experiment from which the binding parameters were also calculated. CD Displacement measurements performed with marker ligands established that the main drug binding sites of HSA are not involved in binding of bixin. Palmitic acid decreased the amplitude of the induced CD bands suggesting a common albumin binding site for bixin and long-chain fatty acids. The above data indicate that HSA plays a significant role in the plasma transportation of bixin and related dietary carboxylic acid carotenoids.     

Multiple ligand-binding properties of the lipocalin member chicken alpha1-acid glycoprotein studied by circular dichroism and electronic absorption spectroscopy: the essential role of the conserved tryptophan residue

Multiple ligand-binding properties of the 30-kDa chicken alpha(1)-acid glycoprotein (cAGP), a member of the lipocalin protein family, were investigated for the first time by using circular dichroism (CD) and UV/Vis absorption spectroscopy methods. By measuring induced CD (ICD) spectra, high-affinity binding (K(a) approximately 10(5)-10(6) M(-1)) of several drugs, dyes and natural compounds to cAGP was demonstrated including antimalarial agents (quinacrine, primaquine), phenotiazines (chlorpromazine, methylene blue), propranolol, non-steroidal antiinflammatory drugs (ketoprofen, diclofenac), tamoxifen, diazepam, tacrine, dicoumarol, cationic dyes (auramine O, thioflavine T, ethidium bromide), benzo[a]pyrene, L-thyroxine, bile pigments (bilirubin, biliverdin), alkaloids (piperine, aristolochic acid), saturated and unsaturated fatty acids. Analysis of the extrinsic CD spectra with the study of the covalently modified protein and CD displacement experiments revealed that a single Trp26 residue of cAGP conserved in the whole lipocalin family is part of the binding site, and it is essentially involved in the ligand-binding process via pi-pi stacking interaction resulting in the appearance of strong induced CD bands due to the non-degenerate intermolecular exciton coupling between the pi-pi* transitions of the stacked indole ring-ligand chromophore. The finding that cAGP is able to accommodate a broad spectrum of ligands belonging to different chemical classes suggests that its core beta-barrel cavity is unusually wide containing overlapping sub-sites. Significance of these new data in understanding of the ligand-binding properties of other lipocalins, especially that of human AGP, and potential practical applications are briefly discussed. Overall, cAGP serves as a simple, ultimate model to extend our knowledge on ligand-binding properties of lipocalins and to study the role of tryptophan residues in molecular recognition processes.     

Importance of pH and disulfide bridges on the structural and binding properties of human α₁-acid glycoprotein

Human α₁-acid glycoprotein (AGP) is an acute phase plasma glycoprotein containing two disulfide bridges. As a member of the lipocalin superfamily, it binds and transports several basic and neutral ligands, but a number of other activities have also been described. Thanks to its binding properties, AGP is also a good candidate for the development of biosensors and affinity chromatography media, and in this context detailed structural information is needed. The structural properties of AGP at different p²Hs and under reducing conditions were analysed by FT-IR spectroscopy. The obtained data indicate that AGP, when denatured, does not aggregate at neutral or basic p²Hs whilst it does at acidic p²Hs. Under reducing conditions the protein is remarkably less thermostable than its oxidized counterpart and presents an enhanced tendency to aggregate, even at neutral p²H. A heat-induced molten globule-like state (MG) was detected at 55 °C at p²H 7.4 and 5.5. At p²H 4.5 the MG occurred at 45 °C with an onset of formation at 40 °C. The MG was not observed under reducing conditions. A lower affinity of chlorpromazine and progesterone for the MG formed at p²H 4.5 and 40 °C was observed, suggesting that ligand(s) may be released near the negative surfaces of biological membranes. Furthermore, the reduced AGP displays an enhanced affinity for progesterone, indicating the importance of disulfide bonds for the binding capacity of AGP.     

Circular dichroism of the peripheral chlorophylls in photosystem II reaction centers revealed by electrochemical oxidation

Visible absorption spectra and circular dichroism (CD) of the red absorption band of isolated photosystem II reaction centers were measured at room temperature during progressive bleaching by electrochemical oxidation, in comparison with aerobic photochemical destruction, and with anaerobic photooxidation in the presence of the artificial electron acceptor silicomolybdate. Initially, selective bleaching of peripheral chlorophylls absorbing at 672 nm was obtained by electrochemical oxidation at +0.9 V, whereas little selectivity was observed at higher potentials. Illumination in the presence of silicomolybdate did not cause a bleaching but a spectral broadening of the 672-nm band was observed, apparently in response to the oxidation of carotene. The 672-nm absorption band is shown to exhibit a positive CD, which accounts for the 674-nm shoulder in CD spectra at low temperature. The origin of this CD is discussed in view of the observation that all CD disappears with the 680-nm absorption band during aerobic photodestruction.     

The 1.8-A crystal structure of alpha1-acid glycoprotein (Orosomucoid) solved by UV RIP reveals the broad drug-binding activity of this human plasma lipocalin

α₁-Acid glycoprotein (AGP) is an important drug-binding protein in human plasma and, as an acute-phase protein, it has a strong influence on pharmacokinetics and pharmacodynamics of many pharmaceuticals. We report the crystal structure of the recombinant unglycosylated human AGP at 1.8 Å resolution, which was solved using the new method of UV-radiation-damage-induced phasing (UV RIP). AGP reveals a typical lipocalin fold comprising an eight-stranded β-barrel. Of the four loops that form the entrance to the ligand-binding site, loop 1, which connects β-strands A and B, is among the longest observed so far and exhibits two full turns of an α-helix. Furthermore, it carries one of the five N-linked glycosylation sites, while a second one occurs underneath the tip of loop 2. The branched, partly hydrophobic, and partly acidic cavity, together with the presumably flexible loop 1 and the two sugar side chains at its entrance, explains the diverse ligand spectrum of AGP, which is known to vary with changes in glycosylation pattern.     

α1-Acid glycoprotein binds human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein viaN-linked glycans

In the present study, we demonstrate a specific low-affinity interaction between recombinant precursor gp160 (rgp160) or surface unit gp 120 (rgp 120) of human immunodeficiency virus type 1 (HIV-1) and ₁-acid glycoprotein (AGP), a human glycoprotein displaying complex typeN-glycans. Binding of rgp 160/rgp 120 to agarose-coupled AGP was dose-dependent, saturable, calcium-, pH- and temperature-dependent. Binding was inhibited by soluble AGP, asialo-AGP, fetuin, -d-GlcNAc₄₇-BSA, -d-Man₂₀-BSA, mannan, complex-type asialo-agalacto-tetraantenary precursor oligosac-charide from human AGP and oligomannose 9 from porcine thyroglobulin; fully deglycosylated AGP was not inhibitory. The three AGP glycoforms separated on immobilized ConA bound rgp 160 to the same extent as did unfractionated AGP. These findings extend our previous results on the carbohydrate-binding properties of HIV-1 envelope (Env) glycoprotein in that they demonstrate the involvement of AGP glycan moieties in the binding to rgp 160/rgp 120. Preincubation of rgp 160 with AGP or mannan significantly reduced its binding to monocyte-derived macrophages (MDM), suggesting that AGP may play a role in preventing binding of soluble or virus-boundEnv glycoprotein to CD4⁺ monocytic cells.     

Hoechst 33258 binds to G-quadruplex in the promoter region of human c-myc

In vitro binding of Hoechst 33258 to the promoter region of human c-myc, d(GG GGAGGG TGG GGA GGG TGG GGA AGG TGG GG) which forms G-quadruplex, both in vitro and in vivo in the presence of metal ions, was investigated by equilibrium absorption, fluorescence, and kinetic surface plasmon resonance methods. Hypochromic effect in UV absorption spectra and blue shift in fluorescence emission maxima of Hoechst in the presence of quadruplex revealed that Hoechst binds to the quadruplex. Analysis of UV and fluorescence titration data revealed that Hoechst binds to quadruplex with binding affinity of the order of 10(6). Anisotropy measurements and higher lifetime obtained from time-resolved decay experiments revealed that quadruplex-bound Hoechst is rotationally restricted in a less polar environment than the bulk buffer medium. From surface plasmon resonance studies, we obtained kinetic association (k(a)) and dissociation (k(d)) of 1.23+/-0.04 x 10(5)M(-1)s(-1) and 0.686+/-0.009 s(-1), respectively. As Hoechst is known to bind A-T-rich region of duplex DNA, here we propose the likelihood of Hoechst interacting with the AAGGT loop of the quadruplex.     

Expression and characterization of a functional canine variant of cytochrome b5 reductase

Cytochrome b5 reductase (cb5r), a member of the flavoprotein transhydrogenase family of oxidoreductase enzymes, catalyzes the transfer of reducing equivalents from the physiological electron donor, NADH, to two molecules of cytochrome b5. We have determined the correct nucleotide sequence for the putative full-length, membrane-associated enzyme from Canis familiaris, and have generated a heterologous expression system for production of a histidine-tagged variant of the soluble, catalytic diaphorase domain, comprising residues I33 to F300. Using a simple two-step chromatographic procedure, the recombinant diaphorase domain has been purified to homogeneity and demonstrated to be a simple flavoprotein with a molecular mass of 31,364 (m/z) that retained both NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities. The recombinant protein contained a full complement of FAD and exhibited absorption and CD spectra comparable to those of a recombinant form of the rat cytochrome b5 reductase diaphorase domain generated using an identical expression system, suggesting similar protein folding. Oxidation-reduction potentiometric titrations yielded a standard midpoint potential (Eo') for the FAD/FADH2 couple of -273+/-5 mV which was identical to the value obtained for the corresponding rat domain. Thermal denaturation studies revealed that the canine domain exhibited stability comparable to that of the rat protein, confirming similar protein conformations. Initial-rate kinetic studies revealed the canine diaphorase domain retained a marked preference for NADH versus NADPH as reducing substrate and exhibited kcat's of 767 and 600 s(-1) for NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities, respectively, with Km's of 7, 8, and 12 microM for NADH, K3Fe(CN)6, and cytochrome b5, respectively. Spectral-binding constants (Ks) determined for a variety of NAD+ analogs indicated the highest and lowest affinities were observed for APAD+ (Ks=71 microM) and PCA+ (Ks=>31 mM), respectively, and indicated the binding contributions of the various portions of the pyridine nucleotide. These results provide the first correct sequence for the full-length, membrane-associated form of C. familiaris cb5r and provide a direct comparison of the enzymes from two phylogenetic sources using identical expression systems that indicate that both enzymes have comparable spectroscopic, kinetic, thermodynamic, and structural properties.     

Measurement of drug-protein binding by immobilized human serum albumin-HPLC and comparison with ultrafiltration

An HPLC method employing CHIRAL-I (150 mm x 3 mm), 5 microm column from Chrom. Tech., immobilized with human serum albumin (HSA), was used to determine in vitro protein binding of several compounds. Experimentally obtained plasma protein data exhibited good correlation with the reported values. The method was compared with the conventional ultra filtration technique and both yielded similar results. Proprietary compounds that could not be analyzed by ultra filtration due to high non-specific binding to filter membrane were successfully analyzed by HSA-HPLC method. On the other hand, two proprietary compounds did not elute from HSA column due to strong binding, but were successfully analyzed by ultra filtration. This proves that both the techniques have their own merits and demerits and should be exploited judiciously as per the requirement. The plasma protein binding studies conducted on four gyrase inhibitors in rat and human plasma exhibited no interspecies difference via ultra filtration method. Further, it was also observed that the protein binding obtained for the four gyrase inhibitors by HSA-HPLC method was not only similar to that obtained by ultra filtration in human plasma but was also in accordance with ex vivo and in vitro protein binding obtained for rat plasma after ultra filtration because these compounds predominantly bind to HSA The binding of several compounds to alpha1-acid glycoprotein (AGP), another important plasma protein, was also examined using AGP immobilized column. However, the data could not be relied upon since some anti-bacterials and non-steroidal anti-inflammatory drugs (NSAIDS), which are known to predominantly bind to HSA, were also found to bind to AGP.     

Immobilization of alpha(1)-acid glycoprotein for chromatographic studies of drug-protein binding

A new method for preparing immobilized alpha1-acid glycoprotein (AGP) for use in drug-protein binding studies was developed and optimized. In this approach, periodate was used under mild conditions to oxidize the carbohydrate chains in AGP for attachment to a hydrazide-activated support. The final conditions chosen for this oxidation involved the reaction of 5.0 mg/mL AGP at 4 degrees C and pH 7.0 with 5-20 mM periodic acid for 10 min. These conditions helped maximize the immobilization of AGP without significantly affecting its activity. This method was evaluated by using it to attach AGP to silica for use in high-performance affinity chromatography and self-competition zonal elution studies. In work with R- and S-propranolol, only one type of binding site was observed for both enantiomers on the immobilized AGP, in agreement with previous studies using soluble AGP. The association equilibrium constants measured for the immobilized AGP with R- and S-propranolol at pH 7.4 and 37 degrees C were 2.7 x 10(6) and 4.2 x 10(6) M(-1), respectively, with linear van't Hoff plots being obtained between 5 and 37 degrees C. Work performed with other drugs also gave good agreement between the behavior seen for immobilized AGP and that for soluble AGP. The same immobilization method described in this work could be used to attach AGP to other materials, such as those used for surface plasmon resonance or alternative biosensors.     

In vitro binding of leukotriene B4 (LTB4) to human serum albumin: evidence from spectroscopic, molecular modeling, and competitive displacement studies

Circular dichroism (CD) and UV absorption spectroscopy were utilized for the first time to investigate the interaction between leukotriene B4 (LTB4) and human serum albumin (HSA) in vitro. The weak intrinsic CD signal of LTB4 was enhanced fivefold in the presence of HSA. The red-shifted, hypochromic, and reduced vibrational fine structure of the ligand/protein UV absorption spectrum indicated complexation of the two molecules in solution. Results obtained from CD titration experiments were subjected to non-linear regression analysis to estimate the binding parameters (Ka = 6.7 x 10(4) M(-1), n = 1). Palmitic acid strongly decreased the induced CD signal of the LTB4/HSA complex, suggesting the role of a high-affinity fatty acid HSA binding site in the leukotriene complexation. Molecular modeling calculations based on the crystal structure of HSA predicted that the long-chain fatty acid site that overlaps with drug binding site II in subdomain IIIA was the most likely binding location for LTB4. Using the drug site II-specific marker ligand rac-ibuprofen, this prediction was confirmed with induced-CD displacement measurements. To the authors' knowledge, the current study represents the first demonstration of binding of LTB4 to HSA in vitro and has implications for the biological transport of this important pro-inflammatory mediator in vivo.     

Biliverdin is the endogenous ligand of human serum alpha1-acid glycoprotein

α₁-Acid glycoprotein (AAG), an acute phase component of the human serum, is a prominent member of the lipocalin family of proteins showing inflammatory/immunomodulatory activities and promiscuous drug binding properties. Both three-dimensional structure of AAG and its precise biological function are still unknown and only a few endogenous AAG ligands have been described to date. CD spectroscopic studies performed with commercial AAG and the separated genetic variants revealed high-affinity binding of biliverdin (BV) and biliverdin dimethyl ester to the ‘F1/S’ fraction of the protein. The preferential accommodation of the right-handed, P-helicity conformers of the pigments by the protein matrix resulted in strong induced CD activity, which was utilized for estimation of the binding parameters and to locate the binding site. It was concluded that both pigments are bound in the central β-barrel cavity of AAG, held principally by hydrophobic interactions. Possible biological implications of the BV binding ability of AAG with special emphasis on the heme oxygenase-1 pathway are discussed.     

In vitro plasma protein binding and aqueous aggregation behavior of astaxanthin dilysinate tetrahydrochloride

The tetrahydrochloride salt of astaxanthin di-L-lysinate (lys(2)AST) is a highly water-dispersible astaxanthin-amino acid conjugate, with an aqueous dispersibility of > or = 181.6 mg/mL. The statistical mixture of stereoisomers has been well characterized as an aqueous-phase superoxide anion scavenger, effective at micromolar (microM) concentrations. In the current study, the aqueous aggregation behavior and in vitro plasma protein binding [with fatty-acid-free human serum albumin (HSA) and alpha(1)-acid glycoprotein (AGP)] were investigated with a suite of techniques, including circular dichroism (CD) and UV-vis spectroscopy, ultrafiltration, competitive ligand displacement, and fluorescence quenching. Induced CD bands obtained in Ringer buffer solution of HSA demonstrated high affinity monomeric binding of the compound at low ligand per protein (L/P) ratios (in aqueous solution alone the carotenoid molecules formed card-pack aggregates). The binding constant ( approximately 10(6)M(-1)) and the binding stoichiometry (approximately 0.2 per albumin molecule) were calculated from CD titration data. CD displacement and ultrafiltration experiments performed with marker ligands of HSA indicated that the ligand binding occurred at a site distinct from the main drug binding sites of HSA (i.e., Sites I and II). At intermediate L/P ratios, both monomeric and aggregated ("chirally complexed") binding occurred simultaneously at distinct sites of the protein. At high L/P ratios, chiral complexation predominantly occurred on the asymmetric protein template. The tentative location of the chirally-complexed aggregation on the HSA template was identified as the large interdomain cleft of HSA, where carotenoid derivatives have been found to bind previously. Only weak binding to AGP was observed. These results suggest that parenteral use of this highly potent, water-dispersible astaxanthin-amino acid conjugate will result in plasma protein association, and plasma protein binding at sites unlikely to displace fatty acids and drugs bound at well-characterized binding sites on the albumin molecule.     

NKG2D and CD94 bind to multimeric α2,3-linked N-acetylneuraminic acid

Killer lectin-like receptors on natural killer cells mediate cytotoxicity through glycans on target cells including the sialyl Lewis X antigen (sLeX). We investigated whether NK group 2D (NKG2D) and CD94 can bind to sialylated N-linked glycans, using recombinant glutathione S-transferase-fused extracellular lectin-like domains of NKG2D (rNKG2Dlec) and CD94 (rCD94lec). Both rNKG2Dlec and rCD94lec bound to plates coated with high-sLeX-expressing transferrin secreted by HepG2 cells (HepTF). The binding of rNKG2Dlec and rCD94lec to HepTF was markedly suppressed by treatment of HepTF with neuraminidase and in the presence of N-acetylneuraminic acid. Moreover, rNKG2Dlec and rCD94lec bound to α2,3-sialylated human α₁-acid glycoprotein (AGP) but not to α2,6-sialylated AGP. Mutagenesis revealed that ¹⁵²Y of NKG2D and ¹⁴⁴F and ¹⁶⁰N of CD94 were critical for HepTF binding. This is the first report that NKG2D and CD94 bind to α2,3-sialylated but not to α2,6-sialylated multi-antennary N-glycans.     

Induced circular dichroism spectra reveal binding of the antiinflammatory curcumin to human alpha1-acid glycoprotein

This paper reports the first experimental evidence on binding of the plant derived curcumin molecule to human alpha1-acid glycoprotein (AGP), an acute phase protein in blood. Oppositely signed induced circular dichroism (CD) bands measured in the visible spectral region in pH7.4 phosphate buffer indicate that the protein binds this natural polyphenol molecule in a left-handed chiral conformation. Decreasing of the intrinsic fluorescence of AGP upon addition of curcumin confirmed the binding to take place. Fluorescence quenching titration curve of AGP allowed to calculate the association constant of the ligand (Ka = 4 x 10(4) M(-1)). Modification of near UV CD spectrum of the protein suggests that curcumin induces changes in the tertiary structure of AGP, which leads to the decrease of binding affinity. By using rac-warfarin and amitriptyline, selective high affinity ligands of F1-S and A genetic variants of AGP, CD displacement experiments showed that curcumin is able to bind to both variants. Molecular docking calculations performed on curcumin-AGP and warfarin-AGP complexes suggest the existence of two alternative binding sites for curcumin; either at the open end of the central hydrophobic cavity or in a surface cleft of the protein.     

The role of protein binding of trivalent arsenicals in arsenic carcinogenesis and toxicity

Three of the most plausible biological theories of arsenic carcinogenesis are protein binding, oxidative stress and altered DNA methylation. This review presents the role of trivalent arsenicals binding to proteins in arsenic carcinogenesis. Using vacuum filtration based receptor dissociation binding techniques, the lifetimes of unidentate (<1s), bidentate (1-2min) and tridentate (1-2h) arsenite containing peptide binding complexes were estimated. According to our experimental data some of the protein targets to which arsenite may bind in vivo include tubulin, poly(ADP-ribose)polymerase (PARP-1), thioredoxin reductase, estrogen receptor-alpha, arsenic(+3)methyltransferase and Keap-1. Arsenite binding to tubulin can lead to several of the genetic effects observed after arsenic exposures (aneuploidy, polyploidy and mitotic arrests). Among many other possible arsenite binding sites are rat hemoglobin, the DNA repair enzyme xeroderma pigmentosum protein A (XPA), and other C2H2, C3H and C4 zinc finger proteins including members of the steroid receptor superfamily (e.g. glucocorticoid receptor). Macromolecules to which arsenite does not bind to include calf thymus DNA, mixed Type II-A histones and bovine H3/H4 histone. Although all six tested arsenicals released iron from ferritin, radioactive arsenite did not bind to the protein horse ferritin.