Small change in structure leads to large difference in protein photocleavage: two porphyrins bearing rhodanine-based pendants

Two 5,10,15,20-tetraphenylporphyrins with one phenyl group anchored to a rhodanine-terminated side chain, RhD-TPP and RhDCOOH-TPP, were designed and synthesized, and their protein photocleavage activities were investigated using bovine serum albumin (BSA) as a model protein. Both porphyrins exhibit similar absorption spectra, fluorescence spectra, fluorescence quantum yields, and singlet oxygen ((1)O(2)) quantum yields in organic solvents due to their structure similarity. They also show similar binding affinities and binding sites toward BSA. However, RhD-TPP is nearly inactive in protein photocleavage while RhDCOOH-TPP can lead to distinct photocleavage of BSA under the same experimental conditions. Such a difference may be attributed to the different binding modes of the two porphyrin derivatives toward BSA, though the apparent binding affinities and the binding sites are similar, and consequently a great difference in the (1)O(2) quantum yields of the two porphyrins bound on BSA. The presence of the COOH group in RhDCOOH is proposed to play an important role, leading to less hydrophobic character and additional interactions towards BSA.     

Copper II complex of a tridentate ligand: an artificial metalloprotease for bovine serum albumin

A copper(II) complex of 2, 6-bis(benzimidazo-2-yl) pyridine was synthesized and its binding properties with bovine serum albumin (BSA) has been evaluated. The binding plot obtained from the absorption titration data gives a binding constant of 2.4 (+/-0.3) x10(3) M(-1). It was found that the charge transfer band of the metal complex was perturbed in the presence of BSA. The gel electrophoresis pattern of BSA incubated with copper(II) complex shows the metalloproteolytic activity of the metal complex. In the presence of oxygen, protein undergoes site-specific cleavage by binding to the histidine residues of domain III, with the resultant formation of four fragments of molecular weight 49, 45, 22 and 17 kDa. This indicates the presence of two specific binding sites in the protein molecule. In the absence of molecular oxygen, the metal complex was found unable to cleave the protein. The circular dichroism (CD) spectrum of the isolated fragments shows nearly 38% and 32% of alpha helical content in 49 and 45 kDa fragments, respectively, which shows that the cleavage leads to no changes in the secondary structure of the protein fragments.     

Copper(II) complex of a tridentate ligand: an artificial metalloprotease for bovine serum albumin

A copper(II) complex of 2, 6-bis(benzimidazo-2-yl) pyridine was synthesized and its binding properties with bovine serum albumin (BSA) has been evaluated. The binding plot obtained from the absorption titration data gives a binding constant of 2.4 (±0.3) ×10³ M⁻¹. It was found that the charge transfer band of the metal complex was perturbed in the presence of BSA. The gel electrophoresis pattern of BSA incubated with copper(II) complex shows the metalloproteolytic activity of the metal complex. In the presence of oxygen, protein undergoes site-specific cleavage by binding to the histidine residues of domain III, with the resultant formation of four fragments of molecular weight 49, 45, 22 and 17 kDa. This indicates the presence of two specific binding sites in the protein molecule. In the absence of molecular oxygen, the metal complex was found unable to cleave the protein. The circular dichroism (CD) spectrum of the isolated fragments shows nearly 38% and 32% of alpha helical content in 49 and 45 kDa fragments, respectively, which shows that the cleavage leads to no changes in the secondary structure of the protein fragments.     

Antimutagenic action of cobaltous chloride on radiation-induced mutations in cultured Chinese hamster cells

The effects of cobaltous chloride on 8-azaguanine (8AG)-resistant mutations induced by gamma-rays or ultraviolet (UV) light in cultured Chinese hamster V79 cells were examined. Cobaltous chloride alone had no significant effects on survival and mutations of V79 cells at concentrations less than 1 x 10(-5) M. Cobaltous chloride at a concentration of 3 x 10(-6) M had a marked effect in reducing 8AG-resistant mutations induced by gamma-rays of 2-6 Gy, when cells were incubated for 6-7 days in the presence of cobaltous chloride after gamma-ray irradiation (posttreatment). The pretreatment of cells with cobaltous chloride for 6 days before gamma-ray irradiation reduced 8AG-resistant mutations induced by gamma-rays. Pre- or post-treatment with cobaltous chloride had no such effect on UV-induced mutations, however. The difference in responsiveness to cobaltous chloride between bacterial and mammalian cell systems is discussed.     

Purification and identification of an estrogen binding protein from rat brain: oligomycin sensitivity-conferring protein (OSCP), a subunit of mitochondrial F0F1-ATP synthase/ATPase

Early studies have suggested the presence in the central nervous system of possible estrogen binding sites/proteins other than classical nuclear estrogen receptors (nER). We report here the isolation and identification of a 23 kDa membrane protein from digitonin-solubilized rat brain mitochondrial fractions that binds 17beta-estradiol conjugated to bovine serum albumin at C-6 position (17beta-E-6-BSA), a ligand that also specifically binds nER. This protein was partially purified using affinity columns coupled with 17beta-E-6-BSA and was recognized by the iodinated 17beta-E-6-BSA (17beta-E-6-[125I]BSA) in a ligand blotting assay. The binding of 17beta-E-6-BSA to this protein was specific for the 17beta-estradiol portion of the conjugate, not BSA. Using N-terminal sequencing and immunoblotting, this 23 kDa protein was identified as the oligomycin-sensitivity conferring protein (OSCP). This protein is a subunit of the FOF1 (F-type) mitochondrial ATP synthase/ATPase required for the coupling of a proton gradient across the F0 sector of the enzyme in the mitochondrial membrane to ATP synthesis in the F1 sector of the enzyme. Studies using recombinant bovine OSCP (rbOSCP) in ligand blotting revealed that rbOSCP bound 17beta-E-6-[125I]BSA with the same specificity as the purified 23 kDa protein. Further, in a ligand binding assay, 17beta-E-6-[125I]BSA also bound rbOSCP and it was displaced by both 17beta-E-6-BSA and 17alpha-E-6-BSA as well as partially by 17beta-estradiol and diethylstilbestrol (DES), but not by BSA. This finding opens up the possibility that estradiol, and probably other compounds with similar structures, in addition to their classical genomic mechanism, may interact with ATP synthase/ATPase by binding to OSCP, and thereby modulating cellular energy metabolism. Current experiments are addressing such an issue.     

Purification and characterization of the human platelet alpha 2-adrenergic receptor

Human platelet alpha 2-adrenergic receptors have been purified approximately 80,000-fold to apparent homogeneity by a five-step chromatographic procedure. The overall yield starting from the membranes is approximately 2%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioiodinated protein from purified receptor preparations shows a single major band of Mr 64,000. The specific binding activity of the alpha 2-adrenergic receptor after four chromatographic steps is 14.5 nmol/mg protein. This value is consistent with the expected theoretical specific activity (15.6 nmol/mg) for a protein with a molecular mass of 64,000 daltons if it is assumed that there is one ligand-binding site/receptor molecule. The purified protein can be covalently labeled with the alkylating alpha-adrenergic ligand, [3H]phenoxybenzamine. This labeling is specific, and it shows that the Mr 64,000 protein contains the ligand binding site of the alpha 2-adrenergic receptor. In addition, the competitive binding of ligands to the purified receptor protein shows the proper alpha 2-adrenergic specificity. The alpha 2-adrenergic receptor contains an essential sulfhydryl residue. Thus, exposure of the purified receptor to the sulfhydryl-specific reagent, phenylmercuric chloride, resulted in an 80% loss of binding activity. This loss of binding activity was prevented when exposure to phenylmercuric chloride was done in the presence of alpha 2-adrenergic ligands, and it was reversed by subsequent exposure to dithiothreitol. Partial proteolysis of purified alpha 2-adrenergic receptors was obtained with Staphylococcus aureus V-8 protease, alpha-chymotrypsin, and papain. In a comparison with purified beta 2-adrenergic receptors, no common partial proteolytic products were found.     

Molecular imprinting of proteins and other macromolecules resulting in new adsorbents

When the model protein bovine serum albumin (BSA) was dissolved in a concentrated aqueous solution of the multifunctional ligand L-malic acid, the solution was lyophilized, and the solid residue thoroughly washed with tetrahydrofuran to extract malic acid, then the resultant ("imprinted") protein was capable of binding 26.4 +/-0.9 mol equivalents of the ligand in anhydrous ethyl acetate. The nonimprinted BSA (i.e., that prepared in the same manner apart from the absence of malic acid) bound less then one-tenth of that amount under identical conditions. Furthermore, both imprinted and nonimprinted BSA exhibited little binding of L-malic acid in water. The imprinted BSA retained its "memory" for the ligand in ethyl acetate even after a prolonged incubation under vacuum; dissolution in water, however, eliminated the imprinted protein's binding capacity. The BSA imprinted with L-malic acid displayed affinity for this ligand not only in ethyl acetate but also in many other anhydrous solvents. It was found that the higher the solvent's propensity to form hydrogen bonds, the lower the protein-ligand binding in it, thus pointing to hydrogen bonds as the driving force of this binding. Studies with completely or partially cleaved BSA, with other globular proteins, glutathione, and poly(L-aspartic acid) revealed that the critical requirement for the imprintability is the presence of a sufficiently long polymeric chain. Moreover, many hydrogen-bond-forming macromolecules other than proteins, such as dextrans and their derivatives, partially hydrolyzed starch, and poly(methacrylic acid), also could be imprinted for subsequent binding in ethyl acetate. The mechanism of imprinting and binding inferred from these experiments involves a multipoint hydrogen bonding in water of each ligand molecule with two or more sites on the polymeric chain, thereby folding a segment of the latter into a cavity around the ligand; following lyophilization and extraction of the ligand, the cavities remain in organic solvents (but not in water) and give rise to ligand binding. This conclusion is supported by the results of binding of numerous malic acid analogs and related ligands to BSA imprinted with L-malic acid. Finally, BSA imprinted with malic acid was used as a selective adsorbent for a chromatographic separation of an equimolar mixture of maleic and acrylic acids in ethyl acetate.     

Characterization of spin-labelled fatty acids and hematoporphyrin binding sites interactions in serum albumin

Electron paramagnetic resonance (EPR) was used to investigate the spin-labelled fatty acid (SLFA) binding equilibrium to human (HSA) and bovine (BSA) serum albumin. The number of 5-doxyl stearate (5-DS) and 16-doxyl stearate (16-DS) binding sites on HSA and BSA were found to be equal, while the association constants, KA values (especially those of the primary binding site) were different. The applied EPR spectra analysis permitting a quantitative distinguishing between slow macromolecular rotation (pi c) and fast anisotropic motion (steric restriction, S) of bound SLFA, allowed SLFA oxazolidinyl ring mobility to be estimated. The 5-DS nitroxide radical is completely immobilized within the HSA protein matrix (S approximately 1.0, pi c approximately 56 +/- 1 ns). The 5-DS when bound to BSA exhibited the presence of more extensive fluctuations (lower S and pi c values) and its immersion depth with respect to BSA surface was calculated to be 4 +/- 2 A. The 16-DS oxazolidinyl radical bound to HSA was found to undergo moderated fluctuations (both S and pi c are smaller with respect to 5-DS) and it is buried deeper within the protein core (rimm = 10 +/- 2 A with respect to BSA surface). The tetrapyrrole ligands hematoporphyrin (Hp) and hematoporphyrin derivative (HpD) were found to induce well detectable changes in the SLFA binding patterns to serum albumin. The action mode was determined to be different for 16-DS (primary) and 5-DS (secondary) serum albumin binding sites: (i) 5-DS is extruded from several binding sites accompanied by an increase in KA in the remaining ones; (ii) simultaneous binding of 16-DS and Hp consists of cooperative and non-cooperative phases (both the number of the independent sites and the parameter of cooperativity, alpha, being dependent on Hp/HSA ratio); (iii) in principal the mobilities of 5-DS and 16-DS bound to HSA are changed, depending on the porphyrin/HSA ratio; and (iv) the effective immersion depth of the paramagnetic centres with respect to the protein surface is increased when Hp is present as a second ligand (rimm = 7 +/- 2 and 16 +/- 2 A for 5-DS and 16-DS, respectively).     

Effect of metal ions on the interaction between bovine serum albumin and berberine chloride extracted from a traditional Chinese Herb coptis chinensis franch

The effect of four metal ions Cu(2+), Ni(2+), Zn(2+) and Co(2+) on the interaction between bovine serum albumin (BSA) and berberine chloride (BC) extracted from a traditional Chinese Herb coptis chinensis franch, was investigated mainly by means of UV and fluorescence spectroscopy in this paper. The four metal ions make the quenching efficacy of BC to BSA higher than that in the absence of these metal ions because of the possible transition of BSA molecular conformation caused by metal ions. It was found that the quenching mechanism is a combination of static quenching with nonradiative energy transfer. In the presence of metal ions, the apparent association constant K(A) and the number of binding sites of BC on BSA are both decreased in a range of 8-19% and 25-28%, respectively, which indicates that the metal ions decrease the binding efficacy of BC on BSA and increase the concentration of free BC simultaneously. The scheme of interaction between BC and BSA in the presence of metal ions is a strong quenching but a weak binding.     

Binding of isofraxidin to bovine serum albumin

The binding of isofraxidin to bovine serum albumin (BSA) was studied under physiological conditions with BSA concentration of 1.5 x 10(-6) mol x L(-1) and drug concentration in the range of 1.67 x 10(-6) mol x L(-1) to 2.0 x 10(-5) mol x L(-1). Fluorescence quenching spectra in combination with uv absorption spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and CD spectroscopy was used to determine the drug-binding mode, binding constant, and the protein structure changes in the presence of isofraxidin in aqueous solution. The linearity of Scatchard plot indicates that isofraxidin binds to a single class of binding sites on BSA and the values given for the binding constants agree very closely with those obtained by the modified Stern-Volmer equation. The thermodynamic parameters, enthalpy change (DeltaH) and entropy change (DeltaS), were calculated to be -17.63 kJ x mol(-1) and 51.38 J x mol(-1) x K(-1) according to the van't Hoff equation, which indicated that hydrophobic interaction played a main role in the binding of isofraxidin to BSA.     

A membrane receptor for gangliosides is associated with central nervous system myelin

A binding protein specific for major neuronal gangliosides was detected on rat brain membranes using a synthetic ganglioside-protein conjugate, 125I-(GT1b)4BSA (bovine serum albumin derivatized with 4 mol of ganglioside GT1b/mol of protein), as a radioligand. Specific binding of the ligand displayed marked regional variation within the brain, with white matter-enriched regions demonstrating the highest binding activity. Autoradiographic localization of 125I-(GT1b)4BSA binding to tissue sections revealed selective association with myelinated pathways throughout the brain. The ligand also bound preferentially to brain subcellular fractions enriched in myelin, even after removal of axolemma. In contrast, peripheral nerve myelin had little binding activity. The myelin-associated ganglioside receptor detected by 125I-(GT1b)4BSA binding appears to be a novel oligodendroglial membrane protein which preferentially recognizes neuronal gangliosides.     

Molecular docking,QSAR properties and DNA/BSA binding,anti-proliferative studies of 6-methoxy benzothiozole imine base and its metal complexes

Abstract

The molecular and QSAR (Quantitative Structure–Activity Relationship) properties of title compound 2-((6-Methoxybenzo[d]thiazol-2-ylimino)methyl)-6-ethoxyphenol (HL) were evaluated employing HyperChem 7.5 tools. The interaction of the 1a–1e complexes of HL with calf thymus DNA (CT-DNA) was investigated by absorption titrations, Fluorescence quenching and viscosity measurements. The experimental data suggest that these complexes bind to CT-DNA through an intercalative mode, wherein DNA-binding affinity of 1e is found to be greater compared to other complexes. The tryptophan emission-quenching with bovine serum albumin (BSA) experiment revealed stronger binding of 1e than other complexes in the hydrophobic region of protein. The photocleavage of plasmid pBR322 DNA investigated in the presence of the title complexes inferred conversion of supercoiled form of DNA plasmid to circular nicked form. Free-radical scavenging activity studies of HL and its metal complexes determined by their interaction with the stable free-radical DPPH have shown promising antioxidant property. Further cytotoxicity studies with HeLa and MCF-7 cell lines indicated that the compounds can efficiently inhibit the cell proliferation in a dose dependent manner. The DAPI staining assay studies revealed the higher potency of 1e to induce apoptosis. Abbreviations BSA Bovine serum albumin protein

CT-DNA Calf thymus DNA

DMSO Dimethyl sulfoxide

DAPI 4′,-6-Diamidino-2-phenylindole dihydrochloride

ESI–MS Electrospray ionization mass spectrometry

IC₅₀ Half-maximal inhibitory concentration

MBTYE 2-((6-methoxybenzo[d]thiazol-2-ylimino) methyl)-6-ethoxyphenol

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

PBS Phosphate-buffered saline

Tris Tris(hydroxymethyl)aminomethane

Communicated by Ramaswamy H. Sarma     

The binding of palmitoyl-CoA to bovine serum albumin

Bovine serum albumin (BSA) is routinely utilized in vitro to prevent the adverse detergent effects of long-chain acyl-CoA esters (i.e., palmitoyl-CoA) in enzyme assays. Determination of substrate saturation kinetics in the presence of albumin would only be valid if the relationship between bound and free substrate concentrations was known. To elucidate the relationship between bound and free palmitoyl-CoA concentrations in the presence of BSA, several different techniques including equilibrium dialysis, equilibrium partitioning, fluorescence polarization and direct fluorescence enhancement were investigated. Direct fluorescence enhancement using a custom synthesized fluorescent probe, 16-(9-anthroyloxy)palmitoyl-CoA (AP-CoA), was the best approach to this question. Measurement of the relationship between mol of palmitoyl-CoA bound per mol of BSA (nu) versus -log[free palmitoyl-CoA] revealed that the binding of palmitoyl-CoA to BSA, like palmitate was nonlinear, suggesting the presence of more than one class of acyl-CoA binding sites. Computer analyses of the binding data gave a best fit to the 2,4 two-class Scatchard model, suggesting the presence of two high-affinity primary binding sites (k1 = (1.55 +/- 0.46) x 10(-6) M-1) and four lower affinity secondary binding sites (k2 = (1.90 +/- 0.09) x 10(-8) M-1). Further analyses using the six parameter stoichiometric (stepwise) ligand binding model supports the existence of six binding sites with the higher affinities associated with the binding of the first mole of palmitoyl-CoA and weaker binding occurring after the first two sites are occupied. The association constants from this model of multiple binding diminish sequentially (i.e., K1 greater than K2 greater than K3 greater than...greater than or equal to K6), suggesting that each mol of long-chain acyl-CoA binds to BSA with decreasing affinities.     

Characterization of Dominant Resistance to Cobalt Chloride in DICTYOSTELIUM DISCOIDEUM and Its Use in Parasexual Genetic Analysis

Strains of Dictyostelium discoideum resistant to cobaltous chloride have been isolated at a frequency of approximately 10^-6. The resistant strains have one of three phenotypes, recessive to wild type, dominant to wild type and dominant to wild type but requiring the presence of cobaltous chloride to maintain resistance. Strains carrying a dominant cobaltous chloride resistance mutation and a recessive growth temperature-sensitive mutation can be mixed with wild-type haploid lines and then subjected to selection so that only diploid lines survive. Differential sensitivity to cycloheximide has also been observed. Hypersensitivity to cycloheximide in combination with dominant cobaltous chloride resistance provides a means of selecting diploids without the use of temperature-sensitive mutations.     

Interactions of some modified mono- and bis-beta-cyclodextrins with bovine serum albumin

Two mono-substituted beta-cyclodextrins and two bridged bis-beta-cyclodextrins, that is, mono(6-(2-aminoethylamino)-6-deoxy)-beta-cyclodextrin (1), mono(6-(2-(2-aminoethylamino)ethylamino)-6-deoxy)-beta-cyclodextrin (2), ethylene-1,2-diamino bis-6-(6-deoxy-beta-cyclodextrin) (3), and iminodiethylene-2,2'-diamino bis-6-(6-deoxy-beta-cyclodextrin) (4), were prepared from beta-cyclodextrin. Their binding ability with bovine serum albumin as a model protein was investigated through proton magnetic resonance (1H NMR), ultraviolet visible spectroscopy (UV-vis), circular dichroism (CD), and fluorescence spectroscopy. In the 1H NMR spectra of the modified cyclodextrins, the resolution of proton signals decreases after the addition of BSA. From the UV and CD spectra, it is found that both the UV absorption and the alpha-helix content of BSA increase with the concentration of the modified cyclodextrins. The protein-ligand interactions cause a fluorescence quenching. The quenching constants are determined using the Stern-Volmer equation to provide an observation of the binding affinity between modified cyclodextrins and BSA. All these results indicate that the modified cyclodextrins can interact with BSA and the bridged bis(beta-cyclodextrin)s (3 and 4) have much stronger interactions than the mono-substituted beta-cyclodextrins (1 and 2). The strong binding stability of bis-cyclodextrins should be attributed to the cooperative effect of two adjacent cyclodextrin moieties. Job's plot shows that the complex stoichiometries of BSA to the modified cyclodextrins were 1:4 for 1 and 2, as well as 1:3 for 3 and 4, respectively.     

Spectroscopic studies on the interaction of riboflavin with bovine serum albumin

The mechanism of interaction of riboflavin (RF) with bovine serum albumin (BSA) using fluorometric and circular dichroism (CD) methods has been reported. The association constant (K) for RF-BSA binding shows that the interaction is non-covalent in nature. Stern-Volmer analysis of fluorescence quenching data shows that the fraction of fluorophore (BSA) accessible to the quencher (RF) is close to unity, indicating that both tryptophan residues of BSA are involved in the interaction. The high magnitude of rate constant for quenching kq (10(13) M(-1) s(-1) indicates that RF binding site is in close proximity to tryptophan residue of BSA. Thermodynamic parameters obtained from data at different temperatures showed that the binding of RF to BSA predominantly involves the formation of hydrophobic bonds. Binding studies in the presence of a hydrophobic probe 8-anilino-1-naphthalene sulphonic acid, sodium salt (ANS) showed that RF and ANS do not share common sites in BSA. The small decrease in critical micellar concentration of anionic surfactant, sodium dodecyl sulphate in the presence of RF shows that ionic character of RF also contributes to binding and is not solubilized inside the micelle. Significant decrease in concentration of free RF has been observed in the presence of paracetamol. The CD spectrum shows the binding of RF leads to a change in the alpha helical structure of BSA.     

Binding of anti-cardiovascular drug to serum albumin: an insight in the light of spectroscopic and computational approaches

Isoprenaline hydrochloride is a potential cardiovascular drug helps in the smooth functioning of the heart muscles. So, we have performed the binding study of ISO with BSA. This study was investigated by UV absorption, fluorescence, synchronous fluorescence, circular dichroism, etc. The analysis of intrinsic fluorescence data showed the low binding affinity of ISO. The binding constant K_b was 2.8 × 103 M-1 and binding stoichiometry (n) was approximately one and the Gibb’s free energy change at 310 K was determined to be -8.69 kcal mol^?1. Negative Gibb’s free energy change shows the spontaneity of the BSA and ISO interaction. We have found ISO-induced alternation in the UV absorption, synchronous fluorescence and CD spectra in the absence and presence of the quencher indicates the complex formation. In synchronous fluorescence, red shift was obtained because of the complex formation of BSA and ISO. The distance (r) between the BSA (donor) and ISO (acceptor) was 2.89 nm, determined by FRET. DLS measurements interpreted complex formation due to the reduction in hydrodynamic radii of the protein in the presence of the drug. The binding site of ISO was found to be nearer to Trp 134 with the help of molecular docking and the ΔG° was found to be –10.2 kcal mol^?1. The esterase activity result suggests that ISO acts as competitive inhibitor. Thus, this study would help to determine the binding capacity of the drug to the protein which may indicate the efficiency of diffusion of ISO into the blood for the treatment of heart diseases.     

The prevention of adsorption of interferents to radiolabelled protein by Tween 20

Radiolabels are often used to quantitatively determine the amount of protein immobilized on chromatographic supports, immunochemical plates and biosensor surfaces. Bovine serum albumin (BSA) was chosen as a model protein for quantitative deposition studies. BSA was radioiodinated (125I-) or fluorescently labelled (fluorescein), then incubated with the following surfaces: quartz, quartz derivatized by 3-aminopropyltriethoxysilane (Qz-APTES), and Qz-APTES reacted with glutaraldehyde or tresyl chloride. The amounts of BSA immobilized to the different surfaces were compared using data from radioactivity and fluorescence assays. Irreproducible results were obtained with radioiodinated BSA due to adsorption/desorption behaviour of an unidentified radioactive species. When the non-ionic detergent Tween 20 was added to the protein/surface incubation mixture, radiolabelled BSA gave reproducible protein binding results which agreed with fluorescent protein binding patterns. The effect of Tween 20 was due to either the binding to BSA displacing the interferent and/or the solubilization of the interferent.     

Structural studies of bovine,equine, and leporine serum albumin complexes with naproxen

Serum albumin, a protein naturally abundant in blood plasma, shows remarkable ligand binding properties of numerous endogenous and exogenous compounds. Most of serum albumin binding sites are able to interact with more than one class of ligands. Determining the protein‐ligand interactions among mammalian serum albumins is essential for understanding the complexity of this transporter. We present three crystal structures of serum albumins in complexes with naproxen (NPS): bovine (BSA‐NPS), equine (ESA‐NPS), and leporine (LSA‐NPS) determined to 2.58 Å (C2), 2.42 Å (P6₁), and 2.73 Å (P2₁2₁2₁) resolutions, respectively. A comparison of the structurally investigated complexes with the analogous complex of human serum albumin (HSA‐NPS) revealed surprising differences in the number and distribution of naproxen binding sites. Bovine and leporine serum albumins possess three NPS binding sites, but ESA has only two. All three complexes of albumins studied here have two common naproxen locations, but BSA and LSA differ in the third NPS binding site. None of these binding sites coincides with the naproxen location in the HSA‐NPS complex, which was obtained in the presence of other ligands besides naproxen. Even small differences in sequences of serum albumins from various species, especially in the area of the binding pockets, influence the affinity and the binding mode of naproxen to this transport protein. Proteins 2014; 82:2199–2208. © 2014 Wiley Periodicals, Inc.     

Dye-binding protein assay using a long-wave-absorbing cyanine probe

A simple and fast protein assay that involves the binding of water-soluble sulfonate heptamethylene cyanine to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 778 to 904 nm, and the increase in absorption at 904 nm is monitored. This assay is very reproducible, of good color stability for at least 80 min, and sensitive at the 100 ng/mL level of human serum albumin (HSA) when a spectrophotometer with near-infrared wavelength is used to measure absorbance. Few chemicals except ionic surfactants such as cetyltrimethylammonium bromide and sodium dodecyl sulfonate interfere with the assay. Purified proteins have different capacities to interact with the dye; under the experimental conditions, the linear ranges of bovine serum albumin (BSA), HSA and gamma-IgG were 200-2000, 100-2400, and 200-3000 ng/mL, respectively. The relative standard deviation for the five replicate determinations of 1200 ng/mL BSA is 2.1%.