Investigation of denaturation of human serum albumin under action of cethyltrimethylammonium bromide by raman spectroscopy

The mechanism of denaturation of human serum albumin (HSA) under action of a cationic detergent—cethyltrimethylammonium bromide (CTAB) is investigated by Raman spectroscopy method. The percentage contents of α-helical segments in polypeptide chain of HSA at denaturation under action of different concentrations of CTAB at different values of pH is determined. It is shown, that more intensive denaturation of HSA under action of CTAB takes place at pH values, larger the isoelectric point of protein (pI 4.7).     

Polarized fluorescence in the study of rotational diffusion of human albumin during denaturation under the action of SDS

Polarized tryptophan fluorescence of human serum albumin (HSA) was analyzed to determine the parameters of rotational diffusion (rotational relaxation time, rotational diffusion coefficient, and the effective Einstein radius) of HSA molecules during denaturation under the action of sodium dodecyl sulfate (SDS). Two stages of HSA denaturation under the action of SDS were shown: (1) loosening of protein globules and (2) unfolding of the amino-acid chain of the protein. HSA denaturation under the action of SDS is a two-stage process at pH values lower than the pI of HAS but passes through stage 1 only at pH values higher than the pI.     

Fluorescence of tryptophan in the denaturation of human serum albumin under the action of sodium dodecyl sulfate

Tryptophan fluorescence of human serum albumin (HSA) when sodium dodecyl sulfate (SDS) was added to a solution of the protein was studied at various pH values, which allowed us to get an idea about the mechanism of the denaturation of HSA under the action of SDS. The two-stage quenching of tryptophan fluorescence of HSA as the concentration of SDS increased was evidence of the two-stage character of denaturation. The first stage involved loosening of protein globules, and the second one was the complete uncoiling of the amino acid protein chain. At solution pH higher than the isoelectric point of the protein (pI 4.7), denaturation stopped at the first stage. At pH values below pI of the protein, the denaturation of the protein under the action of SDS was more effective and deep (involved both stages).     

Denaturation of bovine serum albumin initiated by sodium dodecyl sulfate as monitored via the intrinsic fluorescence of the protein

The tryptophan fluorescence of bovine serum albumin (BSA) is used to study the denaturation transitions in BSA under the influence of sodium dodecyl sulfate (SDS) at various pH values. The stepwise quenching of BSA tryptophan fluorescence and the gradual increase in the degree of anisotropy of BSA tryptophan fluorescence with increasing SDS concentration in solutions indicate the stepwise nature of denaturation: the first stage is a loosening of protein globules, whereas the second is a complete unfolding of the protein amino acid chain. At pH > pI of BSA, the denaturation BSA proceeds through both stages. At pH > pI of BSA, the denaturation BSA runs poorly and stops after the first stage. A more efficient BSA denaturation under the action of SDS occurs at pH < pI of BSA, with the efficiency of BSA denaturation under the influence of SDS decreasing with increasing pH.     

Study of the denaturation of human serum albumin by sodium dodecyl sulfate using the intrinsic fluorescence of albumin

An analysis of the intrinsic tryptophan fluorescence of human serum albumin (HSA) confirms that the denaturation of HSA by sodium dodecyl sulfate takes place in two stages for different pH levels: the first is the disintegration of globules and the second is the complete unfolding of the amino acid chain of HSA. At pH levels below the isoelectric point (pI 4.7) of HSA, denaturation proceeds through both stages, but when the pH is above pI, denaturation ceases in the first stage.     

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.     

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.     

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.     

Phenylalanine fluorescence and phosphorescence used as a probe of conformation for cod parvalbumin

The fluorescence emission and triplet absorption properties of phenylalanine in cod fish parvalbumin type II, a protein that contains no Trp or Tyr, was examined in the time scale ranging from nanoseconds to microseconds at 25°C in aqueous buffer (pH 7.0). In the presence of Ca(II), the decay of fluorescence gave two lifetimes (5.9 and 53 ns) and the triplet lifetime was 425 s. Upon removal of Ca, the fluorescence intensity decreased to values approaching that for free Phe, while the longest fluorescence decay component was 17 ns. At the same time, the decay of triplet showed complex nonexponential kinetics with decay rates faster than in the presence of Ca. Quenching and denaturation analyses suggest that the Phe's are present in a hydrophobic environment in the Ca-bound protein but that the Ca-free protein is relatively unstructured. It is concluded that Phe luminescence in proteins is sensitive to conformation and that the long lifetime of Phe excited states needs to be considered when studying its photochemistry in proteins.     

Characterization of the Binding of Metoprolol Tartrate and Guaifenesin Drugs to Human Serum Albumin and Human Hemoglobin Proteins by Fluorescence and Circular Dichroism Spectroscopy

The interactions of metoprolol tartrate (MPT) and guaifenesin (GF) drugs with human serum albumin (HSA) and human hemoglobin (HMG) proteins at pH?7.4 were studied by fluorescence and circular dichroism (CD) spectroscopy. Drugs quenched the fluorescence spectra of HSA and HMG proteins through a static quenching mechanism. For each protein-drug system, the values of Stern-Volmer quenching constant, bimolecular quenching constant, binding constant and number of binding site on the protein molecules were determined at 288.15, 298.15, 310.15 and 318.15 K. It was found that the binding constants of HSA-MPT and HSA-GF systems were smaller than those of HMG-MPT and HMG-GF systems. For both drugs, the affinity of HMG was much higher than that of HSA. An increase in temperature caused a negative effect on the binding reactions. The number of binding site on blood proteins for MPT and GF drugs was approximately one. Thermodynamic parameters showed that MPT interacted with HSA through electrostatic attraction forces. However, hydrogen bonds and van der Waals forces were the main interaction forces in the formation of HSA-GF, HMG-MPT and HMG-GF complexes. The binding processes between protein and drug molecules were exothermic and spontaneous owing to negative ?H and ?G values, respectively. The values of binding distance between protein and drug molecules were calculated from Förster resonance energy transfer theory. It was found from CD analysis that the bindings of MPT and GF drugs to HSA and HMG proteins altered the secondary structure of HSA and HMG proteins.     

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.     

Optical, Structural and Thermodynamic Studies of the Association of an Anti-leucamic Drug Imatinib Mesylate with Transport Protein

The interaction of an anti-leukemic drug, imatinib mesylate (IMT) with human serum albumin (HSA) was investigated by fluorescence, synchronous fluorescence, three-dimensional fluorescence, circular dichroism and UV–vis absorption techniques under physiological condition. The process of binding of IMT on HSA was observed to be through a spontaneous molecular interaction procedure. IMT effectively quenched the intrinsic fluorescence of HSA via static quenching. The values of binding constant, number of molecules that interact simultaneously with the binding site and thermodynamic parameters were evaluated by carrying out the interactions at three different temperatures. Based on thermodynamic parameters and displacement studies with site probes, it was proposed that the drug bound at Sudlow’s site I of subdomain IIA. The change in the conformation of HSA was evident from synchronous, three-dimensional fluorescence and circular dichroism studies. The distance between the donor (protein) and acceptor (drug) was calculated based on the Foster’s theory of resonance energy stransfer and it was found to be 1.30 nm. The effect of different metal ions on the binding of the drug to protein was also investigated.     

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”.     

Determination of the parameters of the rotational diffusion of complexes of serum albumins with Triton X-100 from analysis of tryptophan fluorescence

The rotational diffusion of complexes of human serum albumin (HSA) and bovine serum albumin (BSA) with neutral surfactant Triton X-100 is study by analyzing the polarized tryptophan fluorescence and its parameters are determined (rotational relaxation time, diffusion coefficient, effective radius). Similarities in the solubilization of both proteins are revealed: an effective solubilization BSA and HSA in solutions containing neutral surfactant Triton X-100 is achieved at concentration of the latter of 0.3 mM, slightly greater than its critical micelle concentration (0.25 mM), with the most significant effect taking place at pH 5.0, a value close to the isoelectric points of the proteins.     

Total Internal Reflection Fluorescence Spectroscopy for Investigating the Adsorption of a Porphyrin at the Glass/Water Interface in the Presence of a Cationic Surfactant Below the Critical Micelle Concentration

Total internal reflection fluorescence (TIRF) spectroscopy was used to investigate the adsorption behavior of meso-tetrakis(p-sulfonatophenyl)porphyrin (TPPS) at the glass/water interface in the presence of a cationic surfactant (cetyltrimethylammonium bromide, CTAB) far below the critical micelle concentration. The adsorption model of TPPS at the glass/water interface in the presence of low concentration of CTAB was proposed, which was different from the adsorption of TPPS in the presence of micelles of CTAB at the glass/water interface. TPPS and CTAB did not form stable complex at the interface in dilute system. The interfacial species of TPPS were analyzed by comparing the spectra of TPPS at the glass/water interface and in the aqueous phase. The influences of the TPPS concentration, the CTAB concentration, and the pH values on the interfacial fluorescence spectra and intensities were studied. It was demonstrated that electrostatic interaction and hydrophobicity performed an important role on the adsorption of TPPS in the presence of CTAB. The different effects of TPPS concentration on the adsorption behaviour of TPPS at different pH were observed for the first time. It was found that the adsorption isotherms of TPPS at glass/water interface could fit Freundlich equation at pH 7.1.     

Complexation of insecticide chlorantraniliprole with human serum albumin: Biophysical aspects

Chlorantraniliprole is a novel insecticide belonging to the diamide class of selective ryanodine receptor agonists. A biophysical study on the binding interaction of a novel diamide insecticide, chlorantraniliprole, with staple in vivo transporter, human serum albumin (HSA) has been investigated utilizing a combination of steady-state and time-resolved fluorescence, circular dichroism (CD), and molecular modeling methods. The interaction of chlorantraniliprole with HSA gives rise to fluorescence quenching through static mechanism, this corroborates the fluorescence lifetime outcomes that the ground state complex formation and the predominant forces in the HSA-chlorantraniliprole conjugate are van der Waals forces and hydrogen bonds, as derived from thermodynamic analysis. The definite binding site of chlorantraniliprole in HSA has been identified from the denaturation of protein, competitive ligand binding, and molecular modeling, subdomain IIIA (Sudlow's site II) was designated to possess high-affinity binding site for chlorantraniliprole. Moreover, using synchronous fluorescence, CD, and three-dimensional fluorescence we testified some degree of HSA structure unfolding upon chlorantraniliprole binding.     

Determining the binding affinity and binding site of bensulfuron-methyl to human serum albumin by quenching of the intrinsic tryptophan fluorescence

Bensulfuron-methyl (BM) is a highly active sulfonylurea herbicide for use on paddy rice. Steady state fluorescence, UV/vis absorption, circular dichroism (CD), time-resolved fluorescence and molecular modeling methods have been exploited to determine the binding affinity and binding site of BM to human serum albumin (HSA). From the synchronous fluorescence, UV/vis, CD and three-dimensional fluorescence spectra, it was evident that the interaction between BM and HSA induced a conformational change in the protein. Steady state and time-resolved fluorescence data illustrates that the fluorescence quenching of HSA by BM was the formation of HSA-BM complex at 1:1 molar ratio. Site marker competitive experiments demonstrated that the binding of BM to HSA primarily took place in subdomain IIIA (Sudlow’s site II), this corroborates the hydrophobic probe ANS displacement and molecular modeling results. Thermodynamic analysis displays hydrophobic, electrostatic and hydrogen bonds interactions are the major acting forces in stabilizing the HSA-BM complex.     

Mn（Ⅱ）,Co（Ⅱ）与HSA相互作用的荧光光谱研究

用荧光光谱法研究了生理ｐＨ和等离子点（ｐＨ＝５．３０）时Ｍｎ（Ⅱ）、Ｃｏ（Ⅱ）与ＨＳＡ的相互作用。根据Ｆｏｒｓｔｅ非辐射能量转移理论,得到了不同ｐＨ时Ｍｎ（Ⅱ）、Ｃｏ（Ⅱ）在ＨＳＡ中的第一强结合位置与Ｔｒｐ－２１４残基间的距离。这一结果远大于文献报道值,根据Ｍｎ（Ⅱ）、Ｃｏ（Ⅱ）在ＨＳＡ中的结合部位及ＨＳＡ的畴结构对这一显著差异进行了讨论。     

A study of the adsorption of the amphiphilic penicillins cloxacillin and dicloxacillin onto human serum albumin using surface tension isotherms

The interaction of human serum albumin (HSA) with two structurally similar anionic amphiphilic penicillins, cloxacillin and dicloxacillin, at 25 °C has been examined by surface tension measurements under conditions at which the HSA molecule was positively (pH 4.5) or negatively charged (pH 7.4). Measurements were at fixed HSA concentrations (0.0125 and 0.125% w/v) and at drug concentrations over a range including, where possible, the critical micelle concentration (cmc). Interaction between anionic drugs and positively charged HSA at pH 7.4 resulted in an increase of the cmc of each drug as a consequence of its removal from solution by adsorption. Limited data for cloxacillin at pH 4.5 indicated an apparent decrease of the cmc in the presence of HSA suggesting a facilitation of the aggregation by association with the protein. Changes in the surface tension-log (drug concentration) plots in the presence of HSA have been discussed in terms of the adsorption of drug at the air-solution and protein-solution interfaces. Standard free energy changes associated with the micellization of both drugs and their adsorption at the air-solution interface have been calculated and compared.     

蛋白质对测定血清中抗精神病类药物的影响及消除

以人血清白蛋白(HSA)为模型,研究了蛋白质对测定血清样品中抗精神病类药物(APDs：安定、盐酸氯丙嗪及奋乃静)的影响。以分子荧光光谱法详细研究了抗精神病类药物与HSA的相互作用,以瑞利散射光谱研究了不同浓度乙醇对蛋白质变性的影响。研究表明：药物与HSA存在牢固结合作用,体积分数为80%的乙醇水溶液为萃取液提取血清样品中的APDs时,可以使血清中蛋白质缓慢变性,充分释放与其结合的药物。乙醇水溶液萃取液中加入K₂HPO₄无机盐后可形成双水相体系,APDs可进入双水相上相实现药物的均相高效萃取,上相经过滤后可直接进行高效液相色谱(HPLC)检测。该方法用于人血清中三种抗精神病类药物的检测,检出限为18.8～38.4 ng·mL-1,回收率为94.2%～98.7%。方法步骤简单,绿色环保,准确。