Determination of the binding constants of nanomarkers of the fluorescein family to human serum albumin

Using three models, the constant of quenching of fluorescence of nanomarkers of the fluorescein family and the actual constants of its binding to human serum albumin (HSA) at different values of pH are determined. The presence of two mechanisms of binding of nanomarkers of the fluorescein family to HSA and anti-cooperativity are shown. The dependence of the constants of the quenching fluorescence of nanomarkers on pH was found: for fluorescein this was nonlinear, for its halogen derivatives (erythsosine, eosin, and Rose Bengal) it was monotonous and decreased with an increase of pH. It is shown that the electronegativity of the atoms in the structural formulas of nanomarkers of the fluorescein family influences the values of the constants of binding of nanomarkers to HSA.     

Fluorescent characteristics of probes of the fluorescein family in human serum albumin solutions

Differences in human serum albumin solutions of fluorescein family probes in their molecular association, in the type of dependence of fluorescence on pH, and in fluorescence anisotropy have been shown. These are due to the electronegativities of the atoms of the lateral radicals in the structural formula and the pK values of the ionizable groups of the probes.     

Polarized fluorescence in investigation of rotational diffusion of the fluorescein family markers in bovine serum albumin solutions

The analysis of polarized fluorescence of the fluorescein family markers was conducted and parameters of their rotational diffusion in bovine serum albumin solutions (BSA) were determined. The degree of fluorescence anisotropy of the markers increases in the BSA solutions, as well as the time of rotational relaxation of the markers, while the rotational-diffusion coefficient of the markers decreases. The differences in the rotational-diffusion parameters between the markers are determined by the values of the electronegativity of the atoms in their structural formulas: the increase of the electronegativity of the atoms in the structural formulas of the markers results in the increase of the degree of fluorescence anisotropy, a decrease of the rotational-diffusion coefficient, and in the increase of the rotational-relaxation time both in the solutions without the protein and with BSA.     

Raman spectroscopy in investigations of secondary structure of human serum albumin at binding of nanomarkers of fluorescein family

The influence of binding of nanomarkers of fluorescein family to HSA on secondary structure of this protein at different values of pH was investigated by Raman spectroscopy method. The greatest changes in secondary structure of HSA, consisting in decreasing of α-helix sites, at binding of fluorescein to HSA occur at pH 5–6. The greatest changes in secondary structure of HSA, consisting in decreasing of α-helix sites, at binding of eosin or erythrosin to HSA take place at values of pH, smaller 5. The differences in changes in secondary structure of HSA at binding of these three nanomarkers are explained by dependences of binding of nanomarkers to HSA on pH which determined by value of electronegativity of atoms of lateral radicals in structural formulas of nanomarkers and, therefore, by value of pK of their ionized groups.     

Ultrasonic spectroscopy in bovine serum albumin solutions

Ultrasonic absorption and velocity spectra in bovine serum albumin (BSA) aqueous solutions have been measured at 20 degrees C over the broad frequency range 0.1-1600 MHz in the pH range 1.5-13.2. Five different techniques were used: the plano-concave resonator, plano-plano resonator, pulse-echo overlap, Bragg reflection, and high-resolution Bragg reflection methods. The absorption spectrum at neutral pH was well fitted to the relaxation curve assuming a distribution of relaxation frequency with a high-frequency cutoff and long low-frequency tail. The relaxation behavior was interpreted in terms of various degrees of hydration of BSA molecules. At acid pH's, excess absorption over that at pH 7 was explained by double relaxation. The pH dependences of the relaxation frequency and maximum absorption per wavelength showed that the relaxation at about 200 kHz was related to the expansion of molecules and that at 2 MHz resulted from the proton transfer reaction of carboxyl group. At alkaline pH's, the excess absorption was explained by triple relaxation. The relaxation at about 200 kHz was associated with a helix-coil transition, and the two relaxations at 2 and 15 MHz were attributed to the proton transfer reactions of phenolic and amino groups, respectively. The rate constants and volume changes associated with these processes were estimated.     

Spectroscopic investigation of binding of three fluorescent nanomarkers to bionanomolecules of human serum albumin in dependence on pH

The analysis of fluorescent characteristics and degree of molecular association of three fluorescent nanomarkers (eosin, erythrosin and fluorescein) in solutions of human serum albumin (HSA) at different pHs is made. The common features for all three nanomarkers under influence of bionanomolecules of HSA are the quenching of the fluorescence, the red shift of maximum of fluorescence and the decrease of degree of molecular association for every fixed value of pH. The differences in dependences of fluorescence and degree of molecular association on pH between fluorescein and its halogen – derivatives (eosin and erythrosin) are registered. It is established that the quenching of fluorescence by HSA is of compound statically–dynamical type. The electronegativity of lateral atoms in structural formulas of nanomarkers forms the basis of explanation of all features of experimental data in the system “fluorescent nanomarker – protein – buffer solution”.     

Structural and functional changes in ultrasonicated bovine serum albumin solutions

Effects of high-intensity ultrasonication on functional and structural properties of aqueous bovine serum albumin (BSA) solutions were investigated. The functional properties of BSA were altered by ultrasonication. Surface activity of BSA increased. Minimal changes were observed in the global structure of BSA but surface charge increased particularly at basic pH values (e.g. pH>9). While dynamic light scattering measurements indicated that the particle size increased up to 3.4 times after 90 min of sonication, no significant increase in the oligomeric state of BSA using blue native PAGE was observed. The amount of free sulfhydryl groups in BSA after 90 min of sonication decreased. The increased particle size and decreased number of free sylfhydryl groups may be attributed to formation of protein aggregates. Surface hydrophobicity increased and circular dichroism spectroscopy and FTIR analysis indicated changes in the secondary structure of BSA. We hypothesize that mechanical, thermal and chemical effects of ultrasonication resulted in structural changes in BSA that altered the functional properties of the macromolecule which may be attributed to the formation of an ultrasonically induced state that differs from a thermally, mechanically or solvent induced state.     

The fluorescence characteristics and the molecular association of the Rose Bengal nanomarker in human serum albumin solutions

The decrease in the degree of molecular association of the Rose Bengal nanomarker in solutions with the addition of human serum albumin (HSA) has been revealed. It has been observed that in solutions with the addition of HSA the fluorescence quenching and the shifting of the fluorescence spectrum peaks of Rose Bengal to the red take place. It has been shown that the dependence of the effective binding constant of binding Rose Bengal to HSA steadily decreases with an increase in the pH value. It has been established that the values of the molecular association degree of Rose Bengal and the values of the effective constant of its binding to HSA depend on the magnitude of the electronegativity of the atoms in its structural formula, as well as on the pK values of its ionizable groups.     

Structure of molecular associates of fluorescent probes in solutions of human serum albumin

Spectral-luminescent characteristics and molecular association processes in solutions of human serum albumin are analyzed at different pH values for three fluorescent probes (eosin, erythrosin, and fluorescein). Common features for all three probes in protein solutions are quenching of the fluorescence, a red shift of the fluorescence maximum, a decrease in the degree of association, and an increase in the angle between dipole moments of dye molecules in dimers. This being so, differences between fluorescein and its halogen derivatives (eosin and erythrosin) are observed in the pH dependences of fluorescence, degree of association, and the angle between dipole moments of probe molecules in dimers. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 75, No. 6, pp. 782–787, November–December, 2008.     

Investigation of the rotational diffusion of the Rose Bengal fluorescent nanomarker in human serum albumin solutions

The polarized fluorescence of the Rose Bengal fluorescent nanomarker in HSA solutions was investigated and parameters of its rotational diffusion were calculated. The increase in the degree of fluorescence polarization, rotational relaxation time, and the effective hydrodynamic radius of Rose Bengal, as well as the decrease of the rotational diffusion coefficient in HSA solutions, were found. The effects of the electronegativity of atoms in the structure of the nanomarker on the parameters of its rotational diffusion were established based on comparison of Rose Bengal with other nanomarkers of the homologous family.     

Ultrasonic absorption of bovine serum albumin solutions in the frequency range 60 to 160 kHz

The ultrasonic absorption of the globular protein bovine serum albumin in aqueous solution has been measured in the frequency range 60 to 160 kHz using a 2-1 spherical resonator. The effect of pH change and of the denaturants urea, guanidine hydrochloride, and sodium dodecyl sulfate on the absorption loss has been studied. It is concluded that a significant ultrasonic absorption process exists which is related to structural helix-coil transition equilibria. For native protein the maximum loss appears to occur at a frequency at least as low as 70 kHz for a pH of about 4.2. This loss process is distinct from those arising from proton-transfer equilibria perturbations which are manifest at pH 3.2 and 11.6 and at peak frequencies of 400 kHz and above.     

Shear-wave detection of structural effects in aqueous solutions of bovine serum albumin and hemoglobin.

Using cylindrical quartz crystal torsional resonators operating at 39 and 75 kHz to generate shear waves in aqueous solutions of the proteins bovine serum albumin and hemoglobin and the polypeptide poly l-glutamic acid, it has been possible to determine the complex dynamic shear viscosities of the solutions. The effects of concentration, pH, and denaturation using various agents have been studied. It is possible to relate the viscosity and configurational elasticity of the solutions, to the intramolecular and intermolecular forces associated with the of the proteins at frequencies between 60 and 400 kHz and attributed to conformational changes of bovine serum albumin and the quaternary doublet interactions of hemoglobin have been confirmed and emphasized by the use of shear waves.     

The effect of lead cations on the fluorescence characteristics of bovine serum albumin in aqueous solution

The effect of lead (heavy metal) cations on the fluorescence characteristics and photophysical parameters (fluorescence intensity and anisotropy, absorption cross section, excited state lifetime, and rates of singlet-triplet conversion and reversible photobleaching) of tryptophan in an aqueous solution of bovine serum albumin (two-tryptophan protein) is studied and compared with the effect in the aqueous solution of tryptophan. It is demonstrated that the effect of lead on the fluorescence characteristics of the protein is manifested at a molar concentration ratio of metal cations and protein macromolecules of greater than 10 and related to the dynamic quenching of the excited state, protein aggregation, and an increase in the rate of singlet-triplet conversion (the effect of a heavy atom) in tryptophan molecules.     

Interaction between varenicline tartrate and bovine serum albumin

This paper mainly investigated the interaction between varenicline tartrate and bovine serum albumin. The Stern–Volmer quenching constant and bimolecular quenching rate constant were determined; furthermore, the fluorescence quenching mechanism between varenicline tartrate and bovine serum albumin was clarified. The binding constants and the number of binding sites were deduced from the double logarithm regression curve. Thermodynamic parameters were calculated, which indicated that the binding process was spontaneous and the acting force were mainly hydrophobic forces. The binding distance was calculated to be 4.80 nm, which means that there was nonradiative energy transfer from varenicline tartrate to bovine serum albumin during the process. And the bovine serum albumin conformation affected by varenicline tartrate was analyzed through ultraviolet–visible and synchronous fluorescence spectroscopy.     

Investigation of the interaction between trazodone hydrochloride and bovine serum albumin

In this paper, the binding of trazodone hydrochloride (TZH) to bovine serum albumin (BSA) was investigated by spectroscopic (fluorescence, spectrophotometry and circular dichroism) techniques under simulative physiological conditions. A strong fluorescence quenching reaction of TZH to BSA was observed and the quenching mechanism was suggested as dynamic quenching according to the Stern-Volmer equation. The binding constants of TZH with BSA at 288, 302 and 309 K were calculated as (1.56±0.003)×10⁴, (2.31±0.002)×10⁴ and (5.44±0.004)×10⁴ M⁻¹, respectively. The thermodynamic parameters, ΔH⁰ and ΔS⁰ were obtained to be 39.86±0.008 kJ mol⁻¹ and 217.89±0.011 J mol⁻¹ K⁻¹, respectively, which indicated the presence of hydrophobic forces between TZH and BSA. The spectral results observed showed that the binding of TZH to BSA induced conformational changes in BSA. Based on the Förster's theory of non-radiation energy transfer, the binding average distance, r between donor (BSA) and acceptor (TZH) was found to be 2.4 nm. The effect of common ions on binding of TZH to BSA was also examined.     

Interaction of gold nanoparticles with bovine serum albumin

The interaction between gold nanoparticles and bovine serum albumin (BSA) in aqueous solutions was studied. The formation of nanoparticle—BSA associates was demonstrated, which is expressed in a bathochromic shift of the surface plasmon resonance band by 5–6 nm in the absorption spectrum. The results were approximated using the Drude model for metal spheres. The thickness of the dielectric (protein) shell of the nanoparticle and its permittivity (refractive index) were calculated.     

Biomimetic synthesis of HgS nanoparticles in the bovine serum albumin solution

HgS nanocrystals conjugated with protein were synthesized in aqueous solution of Bovine Serum Albumin (BSA) at room temperature. The obtained HgS nanoparticles with average diameter about 20–40 nm were characterized by powder X-ray diffraction (XRD), transmission electron microscopy (TEM), selected-area electron diffraction (SAED) and high-resolution transmission electron microscopy (HRTEM). The quantum-confined effect of the HgS nanoparticles is confirmed by the ultraviolet-visible (UV-vis) and photoluminescence (PL) spectrum. The rescults indicate that the BSA not only induce the nucleation, but inhibit the further growth of HgS nanoparticles. The effect of Hg²⁺ on BSA and the change of BSA conformation were studied through Fourier transform infrared (FTIR) spectroscopy and Circular dichroism (CD) spectroscopy. The possible mechanism of HgS nanoparticles growth in the BSA solution was also discussed.     

Laser correlation spectroscopy of the processes of serum albumin denaturation

Denaturation of serum albumin under the influence of temperature and ionic detergent sodium dodecylsulfate (SDS) is investigated by laser correlation spectroscopy of scattered light. It is shown that thermal denaturation is stronger at pH values of the buffer solution of protein that are close to the isoelectric point of the given protein. Coupling of the micelles of SDS with albumin follows the principle of positive cooperativity. The concentration of protein saturation with the surfactant is determined, upon the attainment of which further protein denaturation was not observed. It is shown that the interaction of SDS with albumin is of an electrostatic nature.Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 71, No. 6, pp. 831–835, November–December, 2004.This revised version was published online in April 2005 with a corrected cover date.     

Spectroscopic studies on the interaction of bovine serum albumin with ginkgolic acid: Binding characteristics and structural analysis

The interaction between ginkgolic acid (GA, C15:0) and bovine serum albumin (BSA) is investigated by several spectroscopic methodologies. At first, the binding characteristics of GA and BSA are determined by fluorescence emission spectra. It is showed that GA quenches the fluorescence of BSA and the static quenching constant K_LB is 11.7891×10⁴ L mol^?1 s^?1 at 297 K. GA and BSA form a 1:1 complex with a binding constant of 9.12×10⁵ L mol^?1. GA binds to the Sudlow's drug binding site II in BSA, and the binding distance between them is calculated as 1.63 nm based on the Förster theory. The thermodynamic parameters indicate that the interaction between BSA and GA is driven mainly by hydrophobic forces. On the other hand, structural analysis indicates that GA binding results in an increased hydrophobicity around the tryptophan residues of BSA as revealed by the synchronous fluorescence spectra, and a decrease in α-helix as revealed by the far-UV CD spectra. In addition, ANS, UV–vis and RLS experiments confirmed that GA binding causes a certain structural changes in BSA. These findings provide the binding information between BSA and GA, and may be helpful for pharmacokinetics and the design of dosage forms of GA.