The electrochemistry of proteins and related substances part III. Bovine serum albumin

The electroreduction of bovine serum albumin (BSA) in pH 7.4 solution was studied at the hanging mercury drop electrode by double potential step chronocoulometry and cyclic voltammetry, and at a mercury pool electrode by controlled potential coulometry. The proposed mechanism involves reduction of an adsorbed monolayer of BSA (maximum coverage 3.3 μC^?2); at short times 3 or 4 disulfide bonds are reduced, the product remains adsorbed and may be reoxidized. On the coulometric time scale (hours) 8 or 9 of the total 17 disulfide bonds are broken and an insoluble product which cannot be oxidized is formed.     

Solid-phase luminescent catalyst immunoassay for human serum albumin with hemin as labeling catalyst

A solid-phase luminescent catalyst immunoassay is described for the determination of human serum albumin (HSA) in solution; hemin is used as a label which catalytically amplifies the sensitivity. The method is essentially a non-radioactive and non-enzymatic sandwich immunoassay. Anti-HSA antibody is covalently bound to a transparent plate, which then undergoes the immunochemical reaction with HSA in the test solution, and with the fixed amount of hemin-labeled anti-HSA antibody. After the two-step immunoreaction, the immunochemically-adsorbed hemin-antibody conjugate is quantified by means of the luminescence produced in a solution containing luminol and hydrogen peroxide. The luminescence intensity is correlated with the amount of HSA. The limit of detection for HSA is 1 ng ml^-1.     

Macromolecular interactions of spectinomycin with Bovine serum albumin

Among the pharmacokinetic parameters of chemotherapeutics, serum albumin binding is a critical factor in determining drug distribution and bioavailability. In this study, the binding properties as well as the interaction of spectinomycin with Bovine serum albumin was investigated. Spectinomycin showed stronger binding with BSA at higher temperatures, which diminishes by decreasing the temperature. The binding constant of spectinomycin with BSA varied from 3.1 × 10³ M^?1 at 298 K to 6.3 × 10³ M^?1 at 313 K. By increasing the temperature, from 298 to 313 K, the binding affinity was increased by twofolds. Thermodynamic analysis indicated changes in albumin conformation and partial loss of folding during spectinomycin-albumin binding. The mild-moderate binding affinity of spectinomycin with BSA will be important in determining the drug–drug interactions at the binding sites of BSA. The presence of stronger binding ligand e.g., chloramphenicol, tetracyclines or diclofenac will compete with spectinomycin for its binding sites, therefore, lowering its serum albumin binding. The result of this study will be helpful in understanding of the binding properties and mechanisms of interaction of spectinomycin with bovine serum albumin.     

Bovine serum albumin as a molecular sensor for the discrimination of complex metabolite samples

The potential for using serum albumin (SA) as a broadly applicable molecular sensor was explored in an effort to develop a method for rapid analysis of complex metabolite samples. SA is a protein present at high concentration in blood, which transports a diverse set of compounds including fatty acids, hormones, and drugs. The effectiveness of the bovine ortholog (BSA) as a molecular sensor was tested by analyzing the pool of small molecules bound to the protein after a brief incubation with complex fluids of biological origin. As an initial test, three varietals of red wine were readily distinguished. Further analysis using four varietals of white wine also showed clear separation. In a second analysis using urine, animals in hemorrhagic shock were separated from a group of comparably treated controls. A time course analysis showed that recovery from injury could also be followed using the assay. This finding is significant as there currently is no method or biomarker for predicting the onset of shock. Comparison of samples was based on liquid chromatography mass spectrometry (LCMS) analysis of compounds selectively bound by BSA. Analysis of the samples after protein selection revealed a significant reduction in complexity and clear separation of groups by Principle Component Analysis (PCA). These results show the potential for using cargo-carrying proteins as molecular sensors for screening complex samples without the need for prior knowledge of sample composition or concentration and may streamline elucidation of biomarkers.     

Spectroscopic studies of water-soluble superstructured iron(III) porphyrin. Interaction with the bovine serum albumin protein

Abstract

Acid-base equilibrium of the “one-face”-hindered sulfonated porphyrin, α5,15-[2,2′(dodecamethyleneoxy),(5-sulfonato)diphenyl]-10,20-bis(2-hydroxy,5-sulfonatophenyl)porphyrinato iron(III), has been studied by paramagnetic ¹H NMR. The isotropically shifted signals change in a fast exchange regime on the NMR time-scale. ¹H longitudinal relaxation times and temperature dependence of the chemical shifts were measured and analyzed. The electronic structure of hydroxo specie is characteristic of a six- or five-coordinate high-spin iron(III) porphyrin with an S = 5/2 ground state. The ¹H NMR titration allowed determination of the acidity constant, pK_a 6.2 (0.1 M KNO₃, 25 °C). In addition, we also report the interaction between the monohydroxo iron(III) porphyrin and the bovine serum albumin protein. From a ¹H NMR titration, we have determined the affinity apparent constant, log K_ap 3.2 (pH 7, KNO₃ 0.1 M, 25 °C). The formation of superstructured iron porphyrin-albumin protein adduct was confirmed by electronic absorption spectroscopy and electron paramagnetic resonance.     

Bovine serum albumin unfolding at the air/water interface as studied by dilational surface rheology

Measurements of the surface dilational elasticity close to equilibrium did not indicate significant distinctions in the surface conformation of different forms of bovine serum albumin (BSA) in a broad pH range. At the same time, the protein denaturation in the surface layer under the influence of guanidine hydrochloride led to strong changes in the kinetic dependencies of the dynamic surface elasticity if the denaturant concentration exceeded a critical value. It was shown that the BSA unfolding at the solution surface occurred at lower denaturant concentrations than in the bulk phase. In the former case, the unfolding resulted in the formation of loops and tails at surface pressures above 12 mN/m. The maximal values of the dynamic surface elasticity almost coincided with the corresponding data for the recently investigated solutions of β-lactoglobulin, thereby indicating a similar unfolding mechanism.     

Bovine serum albumin adsorption on nano-rough platinum surfaces studied by QCM-D

The adsorption of bovine serum albumin (BSA) on platinum surfaces with a root-mean-square roughness ranging from 1.49nm to 4.62nm was investigated using quartz crystal microbalance with dissipation (QCM-D). Two different BSA concentrations, 50microg/ml and 1mg/ml, were used, and the adsorption studies were complemented by monitoring the antibody interaction with the adsorbed BSA layer. The adsorption process was significantly influenced by the surface nano-roughness, and it was observed that the surface mass density of the adsorbed BSA layer is enhanced in a non-trivial way with the surface roughness. From a close examination of the energy dissipation vs. frequency shift plot obtained by the QCM-D technique, it was additionally observed that the BSA adsorption on the roughest surface is subject to several distinct adsorption phases revealing the presence of structural changes facilitated by the nano-rough surface morphology during the adsorption process. These changes were in particular noticeable for the adsorption at the low (50microg/ml) BSA concentration. The results confirm that the nano-rough surface morphology has a significant influence on both the BSA mass uptake and the functionality of the resulting protein layer.     

Bovine serum albumin as a means to immobilize DNA on a silver-plated bulk acoustic wave DNA biosensor

A simple, inexpensive, on-line bulk acoustic wave (BAW) DNA biosensor is proposed by using an Ag-plated surface rather than the conventional Au surface. Bovine serum albumin (BSA) is used as an active coating for DNA immobilization. Impedance analysis reveals that the film composed of didodecyl dithiono-oxamide (DDDTO) and BSA can dramatically enhance the amount of immobilized DNA. Detection of dissociation and hybridization of immobilized DNA is demonstrated as an example of the potential application of this type of acoustic wave DNA biosensor in clinical practice.     

Palladium as catalyst in polycondensed matrix, Part III.

In this part we aim at elucidating the chemical state of palladium in the polymer cages. It was established that in samples both the adduct and the polymer exist in the forms of Pd⁰, Pd_ox and metallic Pd. Their ratio depends on the reaction conditions. Conclusions of the three parts are also given.     

Human serum albumin binding and cytotoxicity studies of surfactant-cobalt(III) complex containing 1,10-phenanthroline ligand

The characteristics of the binding reaction of surfactant-cobalt(III) complex, cis-[Co(phen)?(C??H??NH?)]Cl?·3H?O (phen=1,10-phenanthroline, C??H??NH?=tetradecylamine) with human serum albumin (HSA) were studied by fluorescence and UV-vis absorption spectroscopy. In addition, the effect of the surfactant-cobalt(III) complex on the conformation of HSA was analysed using synchronous fluorescence spectroscopy. The experimental results showed that surfactant-cobalt(III) complex caused the fluorescence quenching of HSA through a combination of static and dynamic quenching. The number of binding sites (n) and apparent binding constant (K(a)) of surfactant-cobalt(III) complex (above and below the critical micelle concentration (cmc) were determined at various temperatures. According to the thermodynamic parameters, it is likely that hydrophobic interactions are involved in the binding process. The cancer chemotherapeutic potential of surfactant-cobalt(III) complex on ME-180 cervical cancer cell was determined using MTT assay and specific staining techniques. The complex affected the viability of the cells significantly and the cells succumbed through an apoptosis process as seen in the nuclear morphology and cytoplasmic features. In addition, single-cell electrophoresis indicated DNA damage.     

Conformational changes in human serum albumin induced by sodium perfluorooctanoate in aqueous solutions

Conformational changes in the bulk solution and at the air-aqueous interface of human serum albumin (HSA) induced by changes in concentration of sodium perfluorooctanoate (C(7)F(15)COO(-)Na(+)) were studied by difference spectroscopy, zeta-potential data, and axisymmetric drop shape analysis. zeta-potential was used to monitor the formation of the HSA-C(7)F(15)COO(-)Na(+) complex and the surface charge of the complex. The conformational transition of HSA in the bulk solution was followed as a function of denaturant concentration by absorbance measurements at 280 nm. The data were analyzed to obtain values for the Gibbs energies of the transition in water (DeltaG(0)(W)) and in a hydrophobic environment (DeltaG(0)(hc)) pertaining to saturated protein-surfactant complexes. The conformational changes that surfactants induce in HSA molecules alter its absorption behavior at the air-water interface. Dynamic surface measurements were used to evaluate this behavior. At low [C(7)F(15)COO(-)Na(+)], proteins present three adsorption regimes: induction time, monolayer saturation, and interfacial gelation. When surfactant concentration increases and conformational changes in the bulk solution occur, the adsorption regimes disappear. HSA molecules in an intermediate conformational state migrate to the air-water interface and form a unique monolayer. At high [C(7)F(15)COO(-)Na(+)], the adsorption of denatured molecules exhibits a behavior analogous to that of dilute solutions.     

Conformational change of adsorbed and desorbed bovine serum albumin on nano-sized magnetic particles

Adsorptions of bovine serum albumin (BSA) on nano-sized magnetic particles with and without the presence of carbodiimide were studied. Desorption of BSA from magnetic particles were carried out in either NaOH or Na₂HPO₄ solutions. The structures of native BSA, adsorbed BSA on magnetic particles, and desorbed BSA were studied by several methods, circular dichroism (CD), fluorescence spectroscopy and differential scanning calorimetry (DSC). The magnitude of conformational changes of protein was determined by calculating the α-helix content from the circular dichroism (CD) spectra and by evaluating fluorescence spectrum and DSC thermograms. Adsorbed BSA on magnetic particles shows no thermal transition with respect to the native BSA. The structural change of BSA when desorbed by Na₂HPO₄ solution is much smaller in comparison to that when desorbed by NaOH solution. Hence, this indicates that BSA could be desorbed from nano-sized magnetic particles using Na₂HPO₄ without much conformational change.     

Terbium(III) complex as a luminescent sensor for human serum albumin in aqueous solution

A terbium(III) complex gives off strong luminescence upon reacting with human serum albumin in aqueous solution, which can be used to detect the presence and structural modifications of this protein.