Luminescent spectral characteristics of eosin in solutions of human serum albumin when denatured by treatment with sodium dodecyl sulfate

From analysis of the fluorescence spectra of eosin molecules in a solution with human serum albumin (HSA), we have obtained information about the dynamics of protein conformational rearrangements during denaturing of the protein when treated with sodium dodecyl sulfate (SDS) for different pH values of the solution. We hypothesize that HSA denaturing in the presence of SDS occurs in two stages: the first stage is loosening of the protein globules, and the second stage is complete unfolding of the protein molecules. We have shown that denaturating of the protein in the presence of SDS passes through both stages for a solution pH below the isoelectric point of the albumin, while the denaturing stops in the first stage for a solution pH above the isoelectric point of the albumin. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 73, No. 5, pp. 661–665, September–October, 2006.     

Molecular spectroscopic insight into the binding of batatasin V isomers to human serum albumin

Fluorescence, ultraviolet-visible absorption spectroscopy, circular dichroism spectrum, and molecular docking methods were employed to study the interaction mechanisms of batatasin V and its isomer with human serum albumin. The two isomers both bond reactively to the hydrophobic activity in subdomain IIA, with an approximate binding affinity. Thermodynamic parameters and molecular modeling results manifested that hydrogen bonds and van der Waals force were the main contributors to the interaction. The secondary structure of human serum albumin was altered with the obvious decreased amount of α-helix. The results overall suggested similar binding mechanisms of batatasin V isomers with human serum albumin. This work will promote the further study of batatasins for pharmacological function. It could also help to provide some useful information for further drug design based on batatasins.     

Spectroscopic determination of binding between human serum albumin and a platinum(II) dimethylsulfoxide complex

We have used electronic absorption and fluorescence spectroscopy to study binding between a platinum(II) dimethylsulfoxide complex (cis-[Pt(DMSO)₂Cl₂]) and human serum albumin (HSA), and the effect of complexation on the structure of the protein. We have calculated the binding parameters for binding between cis-[Pt(DMSO)₂Cl₂] and HSA. We have determined the binding constant K_B = (1.2 ± 0.1)·10³ M⁻¹ and the Hill coefficient h = 1.03 ± 0.1. We have determined that binding between cis-[Pt(DMSO)₂Cl₂] and the protein leads to a change in the internal packing of the macromolecule. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 75, No. 4, pp. 573–576, July–August, 2008.     

Interaction of molecules of the neonicotinoid imidacloprid and its structural analogs with human serum albumin

We have used fluorescence spectroscopy methods to show that imidacloprid and its structural analogs form complexes with human serum albumin (HSA). The nature of the spectral changes in the ligand×protein systems and the calculated complexation parameters suggest that these low molecular weight compounds mainly bind to a specific section of the protein molecule, near the tryptophan residue in the 214 position of the polypeptide chain. We have found that the association constants are on the order of 10⁴ M⁻¹, and the affinity of the ligands for HSA varies in the series 6-chloronicotinic acid > 6-methoxynicotinic acid = imidacloprid > the keto analog of imidacloprid. The major contribution to the complexation energy probably comes from hydrophobic interaction forces with participation of the aromatic pyridine ring of the ligands, while additional enhancement of ligand-protein affinity can be provided by the nitroimine group of imidacloprid. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 75, No. 6, pp. 859–866, November–December, 2008.     

Extrinsic fluorescence probe study of human serum albumin using Nile red

Nile red bound to human serum albumin (HSA) shows an order of magnitude increase in the probe's fluorescence intensity. Here, we report on the fluorescence characteristics of the probe-protein complex in Trizma buffer (pH 7.1), urea, guanidine hydrochloride, and AOT/isooctane/buffer reverse micelles using both steady—state and time-resolved fluorescence techniques. With a view to illustrating the use of extrinsic probe fluorescence spectroscopy in protein research, we demonstrate that protein unfolding can be observed through measurements of the probe's time-resolved anisotropy and steady-state fluorescence spectrum. Moreover, this shows that thermal unfolding is fundamentally different from using denaturant, with respect to changes in both the nanosecond diffusional rotation of the probe at intermediate stages and in the denatured protein's structure. Also, the large Stokes shift of Nile red allows the changes in the environment of the probe-protein complex in reverse micelles of varying waterpool size to be easily identified in the steady-state fluorescence. This was not seen in earlier work exploiting the intrinsic tryptophan fluorescence of HSA and further demonstrates the complementary information that extrinsic fluorescence probe studies can offer protein science. We discuss the complex acrylamide quenching characteristics of Nile red bound to HSA in terms of the possibility of at least two binding sites for the probe and the effect of acrylamide on the probe-protein structure at very high quencher concentrations.     

Two distinguishable fluorescent modes of 1-anilino-8-naphthalenesulfonate bound to human albumin

We study the interaction of 1-anilino-8-naphthalenesulfonate (ANS) with human (HSA) and bovine serum albumin (BSA) by phase and modulation fluorescence spectroscopy. We determined that both HSA and BSA show one or two distinguishable fluorescent sites, depending of the ANS/serum albumin ratio. At above a 11 ANS/HSA molar ratio, the steady-state emission spectra for ANS can be resolved in two components: component 1, emitting with a lifetime (₁) of 16 ns and a _1max of 478 nm, with a quantum yield (_f1) of 0.67, and component 2, with a lifetime (₂) of 2–4 ns and a _2max of 483 nm, with an average quantum yield (_f2) of about 0.11. Considering these findings, the binding analysis is fitted with a model of two independent sites. Site 1 has an association constantK _as1=0.87×10⁶M^–1 and a capacity of 1.04 mol of ANS/mol of HSA, and site 2 aK _as2=0.079×10⁶M^–1 and a capacity of 2.34 mol of ANS/mol of HSA. Analysis of fluorescence lifetime distributions shows that the rigidity of the fluorophore environment at site 1 changes when site 2 is occupied. These findings suggest an interconnection between the two sites and that ligands can stabilize the protein's globular structure. To assess the identity of the ANS binding sites we used diazepam as a marker of the site located at the IIIA HSA subdomain and aspirin as a marker of sites located at the IIIA and IIA HSA subdomains. Both ligands displace ANS only from site 1, suggesting that it corresponds to the binding site located at the IIIA sub-domain of the protein. We determined that theK _as values for diazepam and aspirin are 0.113× 10⁶ and 0.021×10⁶ M ^–1 respectively.     

Quinolinesulfonamides: Interaction between bovine serum albumin,molecular docking analysis,and antiproliferative activity against human breast carcinoma cells

The complex formation of bovine serum albumin (BSA) with library of all seven regioisomeric quinolinesulfonamides (QSAs) under physiological condition is studied in this paper. The fluorescence quenching mechanism of BSA by QSAs was discussed and the association constants, as well as the number of binding sites, were calculated. In addition, a molecular docking study of the tested sulfamoylquinolines on the active site of serum albumin is performed. The experimental data and molecular docking studies reveal that sulfamoylquinolines bind in the large hydrophobic cavity of subdomain IB, and in the hydrophobic pockets of BSA subdomains IIA and IIIA by hydrophobic interactions with tryptophanyl (Trp-213) and tyrosyl residues. Moreover, the antiproliferative activity of QSAs against two human breast cancer cell lines (human adenocarcinoma (MCF-7) and human ductal carcinoma (MDA-MB-231)) and a human normal fibroblast is also studied in this paper. The antiproliferative activity of the tested QSAs was comparable to those of cisplatin. The returned data indicate that some of the tested quinolinesulfamoyl derivatives display significant cytotoxic activity.     

Interaction of 1-cyanoethyl-5-chlorouracil with human and bovine serum albumins

The interactions of human (HSA) and bovine (BSA) serum albumins with 1-cyanoethyl-5-chlorouracil (CECU) were investigated by fluorescence spectroscopy, UV absorption spectroscopy, and molecular modeling methods under the simulated physiological conditions. The results of fluorescence measurements indicate that CECU has a strong ability to quench the intrinsic fluorescence of both HSA and BSA through a static quenching procedure. The binding constants (K) at different temperatures and thermodynamic parameters, enthalpy change (ΔH), and entropy change (ΔS) were calculated according to fluorescence data. The results show that hydrophobic interaction is a predominant intermolecular force for stabilizing the complex, which is in agreement with the results of molecular modeling study. The effect of some normal ions on the binding constants is also discussed. Published in Zhurnal Prikladnoi Spektroskopii, Vol. 75, No. 5, pp. 737–745, September–October, 2008.     

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.     

Interaction of nobiletin with human serum albumin studied using optical spectroscopy and molecular modeling methods

The binding of nobiletin to human serum albumin (HSA) was investigated by fluorescence, UV-vis, FT-IR, CD, and molecular modeling. Fluorescence data revealed the presence of a single class of binding site on HSA and its binding constants (K) at four different temperatures (289, 296, 303 and 310 K) were 4.054, 4.769, 5.646 and 7.044×10⁴ M⁻¹, respectively. The enthalpy change (ΔH⁰) and the entropy changes (ΔS⁰) were calculated to be 1.938 kJ mol⁻¹ and 155.195 J mol⁻¹ K⁻¹ according to the Van’t Hoff equation. The binding average distance, r, between the donor (HSA) and the acceptor (nobiletin) was evaluated and found to be 2.33 nm according to the Förster's theory of non-radiation energy transfer. Changes in the CD and FT-IR spectra were observed upon ligand binding along with a significant degree of tryptophan fluorescence quenching on complex formation. Computational mapping of the possible binding sites of nobiletin revealed the molecule to be bound in the large hydrophobic cavity of subdomain IIA.     

Effect of the conditions of isolation on the physicochemical properties of human serum albumin in the norm and with pathology

Differential scanning calorimetry and IR spectrosocopy were used to investigate the effect of the procedure of isolation of human serum albumin on its physicochemical characteristics. It is shown that fractionation of blood plasma with ethylene glycol followed by ion exchange chromatography can be used to obtain albumin of normal donors that is similar to the albumin in the nonfractionated plasma according to melting thermograms. Endotherms of human serum albumin samples that were obtained by affinity chromatography and preparative electrophoresis are bimodal, unlike the monophasic for albumin obtained by polyethylene glycol precipitation. These changes result from a higher content of nonetherified fatty acids in the albumin samples obtained by affinity chromatography and from modification of the secondary protein structure in the samples obtained by electrophoresis. Analysis of melting thermograms of serum albumin from patients with uremia, chronic hepatitis, and peritonitis shows that fractionation of blood with polyethylene glycol preserves the thermodynamic characteristics of the various pathological serum albumins to the greatest extent. The present results demonstrate the advantage of polyethylene glycol fractionation for isolation of native preparations of normal and “pathological” human serum albumin. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 64, No. 1, pp. 32–37, January–February, 1997.     

Nonenzymatic glycosylation of human serum albumin: Fluorescence and chemiluminescence behavior

In the present study the progress of the cross-linking reaction between glyceraldehyde (GCA) and human serum albumin (HSA) was followed by monitoring the development of new absorption bands characteristic of the cross-linked product. Generation of electronically excited species (EES) during horseradish peroxidase (HRP)-catalyzed oxidation of glycosylated HSA was followed by measuring direct chemiluminescence. It was found that adducts of HSA with GCA in the presence of HRP are capable of forming EES. The mechanism of EES generation is discussed further on the basis of fluorescence and chemiluminescence measurements. Formation of EES in studied systems thus could be a possible source of free radicals generatedin vivo during age- and diabetes-related complications.     

Studies on the interaction of gliclazide with bovine serum albumin by fluorescence and synchronous fluorescence spectroscopy

The interaction between gliclazide and bovine serum albumin was investigated by fluorescence and synchronous fluorescence spectroscopy. From the experimental results, it was found that the quenching mechanism was static. The results of the synchronous fluorescence obtained indicated that the binding of gliclazide with bovine serum albumin could affect conformation in bovine serum albumin. In addition, the binding constants (K_a), binding sites (n), thermodynamic parameters, binding forces, Hill’s coefficient, and binding rate of gliclazide to protein calculated from two methods using the same equation were consistent at three different temperatures (298, 310, 318 K). This indicated that as a useful supplement to the conventional method, synchronous fluorescence spectroscopy could be used to study the mechanism of drugs and proteins. The conclusion was verified by UV/vis method.     

Luminescent spectral properties of rhodamine derivatives while binding to serum albumin

We have studied the effect of blood serum albumin on the absorption and fluorescence spectra of rhodamine C (RC), rhodamine 6G (R6G), and rhodamine 3B (R3B). Interaction of the dye with protein is assessed using the binding parameters: binding constants and concentrations of binding sites. We have studied the effect of temperature on the binding parameters. We have observed that heating a mixture of the dye solution with protein for 30 min leads to an increase in the binding constant for rhodamine 3B with protein by a factor of 2, while the concentration of binding sites increases by a factor of 2.3. This is explained by features of the globular protein structure and a change in its conformation when heated. We have shown that rhodamine 3B at a concentration of 10⁻⁵ M is the most effective among the studied rhodamine dyes for application as a fluorescent probe when studying conformational changes in blood serum protein. Report given at the Third International Conference on Liquid State Physics: Current Problems, May 27–31, 2005, Kiev, Ukraine. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 73, No. 3, pp. 380–384, May–June, 2006.     

Development of fluorescent probes for bioimaging applications

Fluorescent probes, which allow visualization of cations such as Ca(2+), Zn(2+) etc., small biomolecules such as nitric oxide (NO) or enzyme activities in living cells by means of fluorescence microscopy, have become indispensable tools for clarifying functions in biological systems. This review deals with the general principles for the design of bioimaging fluorescent probes by modulating the fluorescence properties of fluorophores, employing mechanisms such as acceptor-excited Photoinduced electron Transfer (a-PeT), donor-excited Photoinduced electron Transfer (d-PeT), and spirocyclization, which have been established by our group. The a-PeT and d-PeT mechanisms are widely applicable for the design of bioimaging probes based on many fluorophores and the spirocyclization process is also expected to be useful as a fluorescence off/on switching mechanism. Fluorescence modulation mechanisms are essential for the rational design of novel fluorescence probes for target molecules. Based on these mechanisms, we have developed more than fifty bioimaging probes, of which fourteen are commercially available. The review also describes some applications of the probes developed by our group to in vitro and in vivo systems.     

Complexation of fluoroquinolone antibiotics with human serum albumin: A fluorescence quenching study

Mechanism of interaction and detailed physico-chemical characterization of the binding of four fluoroquinolones: levofloxacin, sparfloxacin, ciprofloxacin HCl and enrofloxacin with human serum albumin has been studied at physiological pH (7.4) using fluorescence spectroscopic technique. The stoichiometry of interaction was found to be 1:1 for all the drugs used. The association constants for the interaction were of the order of 10⁴ in most cases. At low drug:protein ratios, a significant fraction of the added drug was bound. The predominant interactions involved are hydrogen bonding and Van der Waal’s interactions in the case of levofloxacin, hydrophobic interactions in the case of ciprofloxacin hydrochloride and enrofloxacin and hydrogen bonding, hydrophobic and electrostatic interactions in the case of sparfloxacin.The drug binding region did not coincide with that of the hydrophobic probe, 1-anilinonaphthalene-8-sulfonate (ANS). From the displacement of site-specific probes and site-marker drugs, it was concluded that ciprofloxacin hydrochloride is site II-specific while enrofloxacin is a site I-specific drug. Levofloxacin binds at both site I and site II with equal affinity. Sparfloxacin had higher affinity for site II than site I. It is also possible that sparfloxacin binds at the interface between site I and site II. Stern-Volmer analysis of the data showed that the quenching mechanism is predominantly collisional for the binding of ciprofloxacin HCl and enrofloxacin while both static and collisional quenching mechanisms are operative in the case of levofloxacin and sparfloxacin. High magnitude of the rate constant for quenching showed that the process is not entirely diffusion controlled. Circular dichroism (CD) spectroscopic studies showed that the presence of drugs did not cause any major changes in the secondary structure of HSA.     

Studies on interaction between flavonoids and bovine serum albumin by spectral methods

The interaction between three kinds of flavonoids and bovine serum albumin (BSA) was investigated by fluorescence and UV-vis absorption spectrometry. The results indicated that flavonoids have strong ability to quench the intrinsic fluorescence of BSA by forming complexes. The binding constants, number of binding sites, thermodynamic parameters and energy transfer mechanisms were also investigated. Conformation change of BSA was observed from synchronous, three-dimensional fluorescence and circular dichroism spectrum.     

Study on the interaction of La with bovine serum albumin at molecular level

The interaction of La³⁺ to bovine serum albumin (BSA) has been investigated mainly by fluorescence spectra, UV-vis absorption spectra, and circular dichroism (CD) under simulative physiological conditions. Fluorescence data revealed that the quenching mechanism of BSA by La³⁺ was a static quenching process and the binding constant is 1.75×10⁴ L mol⁻¹ and the number of binding sites is 1 at 289 K. The thermodynamic parameters (ΔH=−20.055 kJ mol⁻¹, ΔG=−23.474 kJ mol⁻¹, and ΔS=11.831 J mol⁻¹ K⁻¹) indicate that electrostatic effect between the protein and the La³⁺ is the main binding force. In addition, UV-vis, CD, and synchronous fluorescence results showed that the addition of La³⁺ changed the conformation of BSA.     

Production of uniformly sized serum albumin and dextrose microbubbles

Uniformly-sized preparations with average microbubble (MB) diameters from 1 to 7 μm were produced reliably by sonicating decafluorobutane-saturated solutions of serum albumin and dextrose. Detailed protocols for producing and size-separating the MBs are presented, along with the effects that changing each production parameter (serum albumin concentration, sonication power, sonication time, etc.) had on MB size distribution and acoustic stability. These protocols can be used to produce MBs for experimental applications or serve as templates for developing new protocols that yield MBs with physical and acoustic properties better suited to specific applications. Size stability and ultrasonic performance quality control tests were developed to assure that successive MB preparations perform identically and to distinguish the physical and acoustic properties of identically sized MBs produced with different serum albumin-dextrose formulations and sonication parameters. MBs can be stored at 5 °C for protracted periods (2 weeks to one year depending on formulation).     

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.