Flavin-sensitized Photo-oxidation of Lysozyme and Serum Albumin

The excited state processes of riboflavin, flavin mononucleotide and flavin adenine dinucleotide in argon-saturated aqueous solution were studied in the presence of lysozyme or bovine serum albumin (BSA). UV–Vis absorption and fluorescence spectroscopy indicates that the noncovalent flavin-protein binding is relatively weak. Quenching of the flavin triplet state by BSA, observed by time-resolved photolysis, is less efficient than by lysozyme. Light-induced oxidation of the two proteins and reduction of the three flavins were studied. The quantum yields of the former and latter in the absence of oxygen are up to 0.1 and 0.04, respectively. The effects of pH and sensitizer and protein concentrations were examined in greater detail. The proposed reaction is electron transfer from the tryptophan moiety to the flavin triplet rather than excited singlet state.     

J-aggregation of anionic ethyl meso-thiacarbocyanine dyes induced by binding to proteins

The effects of ribonuclease A (RNase), lysozyme, trypsin, and bovine serum albumin (BSA) on the J-aggregation behavior of 3,3'-bis[sulfopropyl]-5-methoxy-4',5'-benzo-9-ethylthiacarbocyanine (1), 3,3'-bis[sulfopropyl]-4,5,4',5'-dibenzo-9-ethylthiacarbocyanine (2), and 3,3'-bis[sulfopropyl]-5,5'-dimethoxy-9-ethylthiacarbocyanine (3) were studied in aqueous solution. The formation of J-aggregates at pH 6 is induced by RNase for 1-3, by lysozyme for 1 and 2, and by trypsin for 2. The formation of J-aggregates correlates with decay of the dimers and is supported by induced circular dichroism spectra. The concentration of J-aggregates for lysozyme/1 increases with an increase in the protein/dye concentration ratio, reaches a plateau, and then gradually decreases. J-aggregates are characterized by relatively weak fluorescence; e.g., Phi(f) = 0.01 for lysozyme/1, and by a small Stokes shift of 6-8 nm, indicating almost resonance fluorescence. J-aggregation proceeds in the range of seconds to minutes with sigmoidal type kinetic curves for trypsin/2 and nonsigmoidal kinetics in the other cases. The presence of BSA, in contrast to RNase, lysozyme, and trypsin, results in deaggregation of dimers of 1-3 and formation of bound monomers and exhibits intense fluorescence from the trans-monomer; e.g., Phi(f) = 0.22 for BSA/1. Generally, the binding of 1-3 to the proteins is a cooperative process, where the number of binding sites changes from n = 15 for lysozyme/1 to n = 6 for trypsin/2 and n = 0.3 and 1 for BSA/3.     

TRANSIENT SPECIES IN THE PHOTOCHEMISTRY OF EOSIN*

Abstract— Photochemical reactions of eosin in aqueous solution were studied using the flash photolysis technique. In deaerated solution the dye was converted quantitatively to the triplet state during flashing. The triplet dye decayed by first and second order reactions which partly regenerated the dye in the ground state and partly produced semioxidized and semireduced eosin. These radical species were formed in an electron dismutation reaction between two triplet molecules and also in a reaction between one triplet and one unexcited molecule. The radicals recombine rapidly to give the dye in the ground state.
An efficient reversible photooxidation reaction was observed in eosin solutions containing potassium ferricyanide. Semioxidized eosin was formed in high yield by reaction between the triplet dye and the oxidant. The dye was regenerated rapidly in a reverse reaction between the products of the oxidation reaction.
An analogous type of reaction was found to occur in eosin solutions containing p -pheny-lene diamine. This reagent reduced the triplet dye to semireduced eosin; the dye was regenerated in the ground state in a very efficient reverse reaction. The protolytic behaviour of semireduced eosin was studied by varying the pH.
Absorption spectra of the transient products were determined and rate constants for the observed reactions were measured. The results are compared with results from previous studies of fluorescein.     

Characterization of Interaction Between Raltitrexed and Bovine Serum Albumin by Optical Spectroscopic Techniques

The interaction of raltitrexed(RTX) with bovine serum albumin(BSA) was investigated by steady state/lifetime fluorescence spectroscopy and circular dichroism(CD) spectroscopy under the simulative physiological conditions. The results of fluorescence titration reveal that RTX could strongly quench the intrinsic fluorescence of BSA via a static quenching procedure. The obtained binding constant K_A of RTX with BSA was 478630 and 44259 L/mol at 298 and 310 K, respectively. According to van’t Hoff equation, the thermodynamic parameters ΔH, ΔG and ΔS were calculated, indicating that hydrophobic forces were the predominant intermolecular forces in stabilizing the complex. The binding process was a spontaneous process, in which Gibbs free energy change was negative. According to Förster’s non-radioactive energy transfer theory, the distance r between donor(BSA) and acceptor(RTX) was 3.82 nm, suggesting that the energy transfer from BSA to RTX occurred with high probability. Displacement experiment and the number of binding sites calculation confirmed that RTX could bind to the site-I of BSA. Furthermore, the effects of pH and some metal ions on the interaction of RTX with BSA were also investigated. The results of synchronous fluorescence and CD spectra show that the RTX-BSA binding induced conformational changes in BSA.     

Investigation of the interaction between chlorophenols and lysozyme in solution

The binding interactions of lysozyme with 2-chlorophenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol and pentachlorophenol were investigated by UV-vis absorption, CD, fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques under physiological pH 7.40. The binding constants, quenching mechanism, and the number of binding sites were determined by the quenching of lysozyme fluorescence in presence of chlorophenols. H-bonds and hydrophobic interactions played major roles in stabilizing the chlorophenols-lysozyme complex. The distances r between chlorophenols and lysozyme were calculated to be 1.94nm, 2.75nm, 3.54nm, and 3.76nm for 2-CP, 2,4-DCP, 2,4,6-TCP, and PCP, respectively. The effects of chlorophenols on the conformation of lysozyme were analyzed using CD, synchronous fluorescence and three-dimensional fluorescence spectra.     

PHOTODYNAMIC INACTIVATION OF LYSOZYME BY EOSIN

Abstract— It has been demonstrated that singlet oxygen is the major oxidizing entity in the photo-dynamic inactivation of hen egg white lysozyme by eosin, using D₂O to enhance the solvent-induced decay lifetime, and azide ion as a specific scavenger. Two regimes of inactivation can be distinguished depending on whether the sensitizer is free or complexed to the enzyme. The kinetic analysis for free dye sensitization, based on photostationary measurements and inactivation quantum yields, indicates that at least 1 in 15 singlet oxygen interactions with lysozyme leads to loss of lytic activity. The direct attack of triplet eosin makes a lesser overall contribution in air-saturated solutions, where 1 in 4 reactions induces inactivation. Lysozyme binds 1 eosin molecule from pH 4 to 12, leading to almost total quenching of the tryptophyl residue fluorescence without inhibition of the enzymic activity. The inactivation quantum yields indicate that singlet oxygen generated from the bound dye is the inactivating agent, but the dominant attack takes place with the complexed fraction of lysozyme molecules. The tryptophyl residue loss is the same or smaller in changing from H₂O to D₂O despite the 5–10 times increase in quantum yield, indicating that singlet oxygen inactivates also by reacting with residues other than tryptophan. The photochemical and fluorescence results are consistent with the the identification of tryptophyl site 108 with the eosin binding site and a reaction target for singlet oxygen. In a re-examination of earlier work on eosin-sensitized photo-oxidation of I", it has been found that singlet oxygen is the oxidizing agent in aerobic solutions.     

Characterization of the interaction between farrerol and bovine serum albumin by fluorescence and circular dichroism

The binding of farrerol to bovine serum albumin (BSA) in aqueous solution was investigated by fluorescence quenching spectra, synchronous fluorescence spectra, circular dichroism (CD) and the three-dimensional (3D) fluorescence spectra at pH 7.40. The results of fluorescence titration indicated that farrerol could quench the intrinsic fluorescence of BSA in a static quenching way. The cause of showing upward curvy patterns in Stern-Volmer plots was analyzed. The binding sites number n and binding constant K using fluorescence quenching equation at 310 K were calculated. The binding distance and the energy transfer efficiency between farrerol and BSA were also obtained according to the theory of F?rster's non-radiation energy transfer. The effect of some metal ions on the binding constant of farrerol with BSA was also studied. The effect of farrerol on the conformation of BSA was analyzed using CD, synchronous fluorescence spectra and three-dimensional (3D) fluorescence spectra under experimental conditions. Furthermore, the fluorescence displacement experiments indicated that farrerol could bind to the site I of BSA.     

A nonionic surfactant-decorated liquid crystal sensor for sensitive and selective detection of proteins

Proteins are responsible for most biochemical events in human body. It is essential to develop sensitive and selective methods for the detection of proteins. In this study, liquid crystal (LC)-based sensor for highly selective and sensitive detection of lysozyme, concanavalin A (Con A), and bovine serum albumin (BSA) was constructed by utilizing the LC interface decorated with a nonionic surfactant, dodecyl β-d-glucopyranoside. A change of the LC optical images from bright to dark appearance was observed after transferring dodecyl β-d-glucopyranoside onto the aqueous/LC interface due to the formation of stable self-assembled surfactant monolayer, regardless of pH and ion concentrations studied in a wide range. The optical images turned back from dark to bright appearance after addition of lysozyme, Con A and BSA, respectively. Noteworthy is that these proteins can be further distinguished by adding enzyme inhibitors and controlling incubation temperature of the protein solutions based on three different interaction mechanisms between proteins and dodecyl β-d-glucopyranoside, viz. enzymatic hydrolysis, specific saccharide binding, and physical absorption. The LC-based sensor decorated with dodecyl β-d-glucopyranoside shows high sensitivity for protein detection. The limit of detection (LOD) for lysozyme, Con A and BSA reaches around 0.1 μg/mL, 0.01 μg/mL and 0.001 μg/mL, respectively. These results might provide new insights into increasing selectivity and sensitivity of LC-based sensors for the detection of proteins.     

Photosensitizing properties of octacarboxy metallophthalocyanines in aqueous medium and their interaction with bovine serum albumin

Photosensitizing properties of aluminium, silicon, zinc and germanium octacarboxy phthalocyanines ((OH)AlOCPc, (OH)₂SiOCPc, ZnOCPc and (OH)₂GeOCPc) were studied in aqueous medium and in the presence of bovine serum albumin (BSA). Triplet quantum yields increased with increasing atomic number of the central metals of the metallophthalocyanine. The efficiency of singlet oxygen generation via energy transfer from the excited triplet state of the octacarboxy metallophthalocyanines (MOCPcs) to ground state oxygen increased markedly in the presence of BSA. The triplet state lifetimes of the MOCPc complexes in the presence of BSA were found to be longer than in the absence of BSA, ranging from 110 to 580 μs. These complexes bind readily to BSA. Stern–Volmer quenching constant K_SV as well as the binding constant k_b values were calculated. The probable mechanism of quenching of BSA fluorescence by the MOCPc complexes is by static quenching.     

An electrospray ionization mass spectrometry investigation of 1-anilino-8-naphthalene-sulfonate (ANS) binding to proteins

The binding of 1-anilino-8-naphthalene-sulfonic acid (ANS) to various globular proteins at acidic pH has been investigated by electrospray ionization mass spectrometry (ESI-MS). Maximal ANS binding is observed in the pH range 3-5. As many as seven species of dye-bound complexes are detected for myoglobin. Similar studies were carried out with cytochrome c, carbonic anhydrase, triosephosphate isomerase, lysozyme, alpha-lactalbumin, and bovine pancreatic trypsin inhibitor (BPTI). Strong ANS binding was observed wherever molten globule states were postulated in solution. ANS binding is not observed for lysozyme and BPTI, which have tightly folded structures in the native form. Alpha-lactalbumin, which is structurally related to lysozyme but forms a molten globule at acidic pH, exhibited ANS binding. Reduction of disulfide bonds in these proteins leads to the detection of ANS binding even at neutral pH. Binding was suppressed at very low pH (<2.5), presumably due to neutralization of the charge on the sulfonate moiety. The distribution of the relative intensities of the protein bound ANS species varies with the charge state, suggesting heterogeneity of gas phase conformations. The binding strength of these complexes was qualitatively estimated by dissociating them using enhanced nozzle skimmer potentials. The skimmer voltages also affected the lower and higher charge states of these complexes in a different manner.     

Spectroscopic Investigation of the Interactions of Cryptotanshinone and Icariin with Two Serum Albumins

The interactions of two drugs, cryptotanshinone (CTS) and icariin, with bovine serum albumin (BSA) and human serum albumin (HSA) have been investigated using multiple spectroscopic techniques under imitated physiological conditions. CTS and icariin can quench the fluorescence intensity of BSA/HSA by a static quenching mechanism with complex formation. The binding constants of CTS–BSA, CTS–HSA, icariin–BSA and icariin–HSA complexes were observed to be 1.67 × 10⁴, 4.04 × 10⁴, 4.52 × 10⁵ and 4.20 × 10⁵ L·mol^?1, respectively at 298.15 K. The displacement experiments suggested icariin/CTS are primarily bound to tryptophan residues of the proteins within site I and site II. The thermodynamic parameters calculated on the basis of the temperature dependence of the binding constants revealed that the binding of CTS–BSA/HSA mainly depends on van der Waals interaction and hydrogen bonds, and yet the binding of icariin–HSA/BSA strongly relies on the hydrophobic interactions. The binding distances between BSA/HSA and CTS/icariin were evaluated by the Föster non-radiative energy transfer theory. The results of synchronous fluorescence, 3D fluorescence, FT-IR and CD spectra indicates that the conformations of proteins were altered with the addition of CTS or icariin. In addition, the effects of some common ions on the binding constants of CTS/icariin to proteins are also discussed.     

LASER FLASH PHOTOLYSIS OF EOSIN AND ITS COMPLEX WITH LYSOZYME

Abstract— The transient absorption spectra of aqueous solutions of eosin and of the lysozyme-eosin complex have been examined after excitation with a Q -switched frequency-doubled (347 nm) ruby laser pulse. Eosin itself gives three broad, intense short-lived absorption bands with maxima at wavelengths of 410, 460 and 580 nm, which other workers have identified with the semi-reduced and semi-oxidized radicals and the triplet state of the dye, respectively. In the complex with lysozyme, the yield and lifetime of the eosin triplet are greatly reduced in comparison with the free dye. It is suggested that excited eosin, when bound to lysozyme, decays mainly from the singlet state by pathways such as charge transfer which are not available to the free molecule.     

Biomimetic interactions of proteins with functionalized cadmium sulfide quantum dots

Interactions between various modified semiconductor nanocrystal, cadmium sulfide quantum dots (CdS QDs) and bovine serum albumin (BSA) and lysozyme (LZY) were investigated. CdS QDs capped with mercaptoethanol (MPA), l-cysteine (Lcys) and glutathione (GSH) were synthesized in aqueous solution and characterized by UV-vis and fluorescence spectrum. Circular dichroism (CD) and fluorescence spectrum were used to detect the interactions between as-prepared CdS QDs and protein molecules. The interaction parameters, including binding constant (K_b), binding site number (n) and quench constant (K_q), were determined by fluorescence spectrum. The changes of secondary structures of the proteins were detected by CD. The results imply that CdS QDs modified by different agents have dramatically different binding strength with protein molecules. The results obtained here analyze the biosafety of CdS QDs in terms of the biological behavior of biomolecules and could serve as basis for the application of CdS QDs to bioscience.     

Microcalorimetric study of the adsorption of PEGylated lysozyme on a strong cation exchange resin

Adsorption of native as well as mono-, di-, and tri-PEGylated lysozyme on Toyopearl Gigacap S-650M in sodium phosphate buffer is studied by isothermal titration calorimetry and by independent adsorption equilibrium measurements at pH 6 and 25°C. The production and separation of PEGylated lysozyme is discussed. Two different PEG sizes are used (5 kDa and 10 kDa) which leads to six different forms of PEGylated lysozyme which were systematically studied. The sodium chloride concentration is varied according to the elution conditions in the production process. The specific enthalpy of adsorption Δh(p)(ads) is determined from the calorimetric and the adsorption equilibrium data. It was found to be exothermal and constant with increasing adsorber loading for native lysozyme. For all PEGylated forms there is an influence of the adsorber loading on Δh(p)(ads) which is found to become more important with increasing PEGylation degree (total molecular weight of conjugated PEG). At low loadings the adsorption of all PEGylated lysozyme forms is exothermal. With increasing loading the adsorption becomes less exothermal and for the species with higher PEGylation degree also endothermal effects are observed at higher loadings. A thermodynamic analysis is carried out by which the enthalpic and entropic contributions to the binding constants are quantified. The findings are discussed on a molecular level. The results provide insight into the adsorption mechanisms of polymer-modified proteins on chromatographic resins.     

光谱法测定伊曲康唑与牛血清和人血清白蛋白相互作用

用荧光光谱和紫外吸收光谱法, 在pH=7.4±0.1的0.1 mol·L-1磷酸缓冲溶液中, 研究了伊曲康唑与牛血清白蛋白(BSA)和人血清白蛋白(HSA)的相互作用. 实验结果表明, 伊曲康唑与牛血清白蛋白和人血清白蛋白作用的猝灭常数均随着温度的升高而降低, 伊曲康唑可以有规律地使血清白蛋白内源荧光猝灭, 其猝灭机理可认为是伊曲康唑与白蛋白形成复合物的静态猝灭. 获得了在不同温度下, 伊曲康唑与血清白蛋白作用的结合常数以及△G、△H和△S等热力学参数. 根据所得结果可推断伊曲康唑与白蛋白的作用力主要为疏水作用力, 同时, 利用荧光共振能量转移理论(FRET)计算得出了伊曲康唑与白蛋白结合位置的距离d. 而且, 利用同步荧光光谱和紫外光谱揭示了该反应中蛋白的结构和其微环境的变化.     

Probing the Binding Interaction of a Phenazinium Dye with Serum Transport Proteins: A Combined Fluorometric and Circular Dichroism Study

In the present investigation, an attempt has been made to study the interaction of phenosafranin (PSF), a cationic phenazinium dye with the transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA), employing steady-state and time-resolved fluorometric and circular dichroism (CD) techniques. The photophysical properties of the dye are altered on binding with the serum proteins. An explicit study with respect to the modification of the fluorescence and fluorescence anisotropy upon binding, effect of denaturant, fluorescence lifetime and CD measurements reveal that the dye binds to both BSA and HSA with almost the same affinity. Far-UV CD spectra indicate a decrease in the percentage of α-helicity only for BSA upon binding with the probe. Near-UV CD responses indicate an alteration in the tertiary structure of both the transport proteins because of binding.     

Characterization of a planar poly(acrylic acid) brush as a materials coating for controlled protein immobilization

The adsorption of two different proteins at a planar poly(acrylic acid) (PAA) brush was studied as a function of the ionic strength of the protein solutions applying total internal reflection fluorescence (TIRF) spectroscopy. Planar PAA brushes were prepared with a grafting density of 0.11 nm(-2) and were characterized using X-ray reflectometry. Hen egg-white lysozyme and bovine serum albumin (BSA) were used as model proteins, which have a net positive and negative charge at neutral pH-values, respectively. It has been found that both proteins adsorb strongly at a planar PAA brush at low ionic strength. Whereas lysozyme interacts with a PAA brush under electrostatic attraction at neutral pH-values, BSA binds under electrostatic repulsion at pH > 5. Even at pH = 8, significant amounts of BSA are adsorbed to a planar PAA brush. In addition, the reversibility of BSA adsorption has been characterized. Dilution of a BSA solution leads to an almost complete desorption of BSA from a PAA brush at short contact times. When the ionic strength of the protein solutions is increased to about 100-200 mM, a planar PAA brush appears largely protein-resistant, regardless of the protein net charge. The results of this study indicate that the salt-dependent protein affinity of a PAA brush represents a unique effect that must be explained by a novel protein-binding mechanism. On the basis of a recent model, it is suggested that a release of counterions is the most probable driving force for protein adsorption at a PAA brush. In a general view, this study characterizes a planar PAA brush as a new materials coating for the controlled immobilization of proteins whose use in biotechnological applications appears to be rewarding.     

Binding behavior of DEHP to albumin: spectroscopic investigation

Bis(2-ethylhexyl)phthalate (DEHP) is one of the biggest selling synthetic plasticizers which can migrate to environment and enter human body via air, water, medical apparatus, and food. In this paper, three-dimensional fluorescence (3D-FL) spectroscopy, UV–visible absorption spectroscopy and circular dichroism (CD) spectroscopy were employed to explore the binding of DEHP to bovine serum albumin (BSA) at the physiological conditions. The number of binding sites n and observed binding constant K _b was measured by fluorescence quenching method. It was found that the fluorescence quenching was static quenching mechanism and caused by the formation of DEHP–BSA complex at ground state. The enthalpy change (ΔH ^θ), Gibbs free energy change (ΔG ^θ) and entropy change (ΔS ^θ) were calculated at four different temperatures. Site marker competitive displacement experiments were carried out to identify the binding location. The results demonstrated that DEHP bound primarily on Sudlow’s site I in domain IIA of BSA molecule. The distance r (2.95?nm) between donor (BSA) and acceptor (DEHP) was obtained based on F?rster’s non-radiation fluorescence resonance energy transfer (FRET) theory. Furthermore, the CD spectral results indicated that the secondary structure of BSA changed in presence of high concentration of DEHP, which implied that high level of DEHP in plasma was potentially poisonous. The study is helpful to evaluating the health risk of DEHP and understanding its functional effects on protein during the blood transportation process.     

Study on the binding of luteolin to bovine serum albumin

Binding of luteolin (LU) to bovine serum albumin (BSA) was investigated at 298, 308 and 318K at pH 7.4 using spectrophotometric techniques such as fluorescence emission, circular dichroism (CD). The data obtained from fluorescence quenching experiments showed that LU was bound to BSA and binding constants and the number of binding sites (n approximately 1) were obtained. The thermodynamic parameters DeltaH(0), DeltaS(0), DeltaG(0) at different temperatures were calculated. They indicated that both hydrophobic forces and hydrogen bonds are the major interactions between LU and BSA. A value of 3.12nm for the average distance r between LU (acceptor) and tryptophan residue (Trp) of BSA (donor) was derived from the fluorescence resonance energy transfer. The effects of some common metal ions on the binding are also considered. Besides, the interaction of BSA with LU led to a change in the conformation of BSA.     

绞股蓝皂苷与牛血清白蛋白相互作用的荧光光谱研究

用荧光光谱技术研究了绞股蓝皂苷与牛血清白蛋白(BSA)在pH=7.40的Tris-HCl缓冲溶液中的相互作用;通过计算确定了绞股蓝皂苷与BSA的结合位点数和结合常数,利用热力学分析探讨了绞股蓝皂苷与BSA之间的结合方式;同时采用同步荧光技术考察了绞股蓝皂苷对BSA构象的影响.结果表明,绞股蓝皂苷对牛血清白蛋白的荧光猝灭过程为静态猝灭;二者主要靠疏水作用和静电引力结合.