Underlying molecular interaction of bovine serum albumin and linezolid: a biophysical outlook
25 Jan 2018-Journal of Biomolecular Structure & Dynamics (Taylor & Francis)-Vol. 36, Iss: 2, pp 387-397
TL;DR: It is concluded that linezolid interacts with BSA in 1:1 ratio through hydrophobic, hydrogen bonding and ionic interactions, and this may not affect the secondary structure of the protein.
Abstract: Linezolid, one of the reserve antibiotic of oxazolidinone class has wide range of antimicrobial activity. Here we have conducted a fundamental study concerning the dynamics of its interaction with bovine serum albumin (BSA), and the post binding modification of the later by employing different spectroscopic (absorption, fluorescence and circular dichroism (CD) spectroscopy) and molecular docking tools. Gradual quenching of the tryptophan (Trp) fluorescence upon addition of linezolid to BSA confirms their interaction. Analysis of fluorescence quenching at different temperature indicates that the interaction is made by static complex formation and the BSA has one binding site for the drug. The negative Gibbs energy change (ΔG0), and positive values of enthalpy change (ΔH0) and entropy change (ΔS0) strongly suggest that it is an entropy driven spontaneous and endothermic reaction. The reaction involves hydrophobic pocket of the protein, which is further stabilized by hydrogen bonding and electrostatic intera...
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TL;DR: The interaction of Pyrogallol with human serum albumin (HSA) was investigated by UV, fluorescence, Circular dichroism, and molecular docking methods and indicated that PG induced conformational changes in the structure of HSA.
Abstract: In the present study, the interaction of Pyrogallol (PG) with human serum albumin (HSA) was investigated by UV, fluorescence, Circular dichroism (CD), and molecular docking methods. The results of ...
29 citations
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TL;DR: The collaborative results of spectroscopic studies indicated that the microenvironment and the conformation of HSA and BSA were significantly perturbed upon interaction with complex [C36H50N8O6Cu].
Abstract: The interaction studies of CuII nalidixic acid–DACH chemotherapeutic drug entity, [C36H50N8O6Cu] with serum albumin proteins, viz., human serum albumin (HSA) and bovine serum albumin (BSA) employin...
26 citations
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TL;DR: Olmutinib (HM61713, OMT), a novel third-generation tyrosine kinase inhibitor with good orally absorption, is able to inhibit selectively epidermal growth factor receptor mutations and can provide valuable reference for designing new anti-tumor drugs.
Abstract: Olmutinib (HM61713, OMT), a novel third-generation tyrosine kinase inhibitor with good orally absorption, is able to inhibit selectively epidermal growth factor receptor mutations. In this work, the binding behavior of OMT with model transport protein bovine serum albumin (BSA) was assessed with the help of spectroscopic and molecular docking approaches. A fluorescence quenching of BSA was observed during the binding interaction of OMT with BSA, followed by a static mechanism was demonstrated. The affinity of OMT with BSA was moderate due to the binding constant of 2.75 × 105 M−1 at 293 K. The hydrophobic interactions, hydrogen bonds as well as van der Waals forces were acting as the predominant contributions in the OMT-BSA complexation process. The findings from site competitive experiments revealed that OMT tended to bind to subdomain IB (site III) of BSA, which was in keep with the outcomes of molecular docking studies. Based on synchronous and 3D fluorescence studies, we found that the conformation of BSA was changed owing to the binding with OMT. The FT-IR and UV-Vis studies further corroborated that the binding of OMT to BSA changed the secondary structure of BSA. Additionally, the experimental data showed that some metal ions (e.g. Ni2+, Fe3+, Mg2+, Cu2+) could promote the binding interaction of OMT with BSA. This study is beneficial to further assess the pharmacological properties of OMT and can provide valuable reference for designing new anti-tumor drugs.
23 citations
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TL;DR: In vitro cytotoxicity of triamterene against HCT116 and CT26 cells showed promising anticancer effects and viscosity data confirmed that triamTerene binds to calf–thymus DNA through intercalation binding mode.
Abstract: The anticancer activity of triamterene on HCT116 and CT26 colon cancer cells lines was investigated Furthermore, the mechanism of interaction between triamterene and calf thymus DNA (ct-DNA) and a
23 citations
Cites methods from "Underlying molecular interaction of..."
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TL;DR: In this article , the interaction between BRT and bovine serum albumin (BSA) was studied by fluorescence spectroscopy, UV spectrophotometry, and molecular docking approaches.
Abstract: Baricitinib (BRT) is an orally administered small molecule JAK inhibitor, which mainly inhibits JAK-1 and JAK-2. And it is used to treat autoimmune diseases such as rheumatoid arthritis (RA), atopic dermatitis and systemic lupus erythematosus. In this paper, the interaction between BRT and bovine serum albumin (BSA) was studied by fluorescence spectroscopy, UV spectrophotometry, fourier transform infrared spectroscopy (FT-IR) and molecular docking approaches. It has been proved that the fluorescence quenching mechanism of BSA in the mutual complexation process of BRT and BSA was a static quenching. The number of binding sites (n) of BRT-BSA complex was about 1. The binding site was located at site II of BSA and its binding constant was about 5.01 × 103 M−1 at 298 k. It is indicated that there was a weak binding between BRT and BSA. By analyzing the sign and magnitude of the free energy change (ΔG0), enthalpy change (ΔH0) and entropy change (ΔS0), it can be found that the complexation process of BRT and BSA is spontaneous and enthalpy-driven. Van der Waals forces and hydrogen bonds play a critical part in the complexation process of BRT with BSA. Based on synchronous fluorescence and three-dimensional fluorescence, it was found that the microenvironment around the residues of Tyr and Trp of BSA had no significant change after binding to BRT. FT-IR and molecular docking studies confirmed that the secondary structure of protein changed during the complexation of BRT with BSA. Experimental results showed that the addition of some metal ions (Ca2+, Zn2+, Cu2+ and Co2+) significantly increased the binding interaction of BRT with BSA. This study is helpful to further evaluate the pharmacological characteristics of BRT and provides an important reference for the design of new drugs.
19 citations
References
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01 Jan 1983
TL;DR: This book describes the fundamental aspects of fluorescence, the biochemical applications of this methodology, and the instrumentation used in fluorescence spectroscopy.
Abstract: Fluorescence methods are being used increasingly in biochemical, medical, and chemical research. This is because of the inherent sensitivity of this technique. and the favorable time scale of the phenomenon of fluorescence. 8 Fluorescence emission occurs about 10- sec (10 nsec) after light absorp tion. During this period of time a wide range of molecular processes can occur, and these can effect the spectral characteristics of the fluorescent compound. This combination of sensitivity and a favorable time scale allows fluorescence methods to be generally useful for studies of proteins and membranes and their interactions with other macromolecules. This book describes the fundamental aspects of fluorescence. and the biochemical applications of this methodology. Each chapter starts with the -theoreticalbasis of each phenomenon of fluorescence, followed by examples which illustrate the use of the phenomenon in the study of biochemical problems. The book contains numerous figures. It is felt that such graphical presentations contribute to pleasurable reading and increased understand ing. Separate chapters are devoted to fluorescence polarization, lifetimes, quenching, energy transfer, solvent effects, and excited state reactions. To enhance the usefulness of this work as a textbook, problems are included which illustrate the concepts described in each chapter. Furthermore, a separate chapter is devoted to the instrumentation used in fluorescence spectroscopy. This chapter will be especially valuable for those perform ing or contemplating fluorescence measurements. Such measurements are easily compromised by failure to consider a number of simple principles."
27,352 citations
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TL;DR: On the basis of the thermochemical behavior of small molecule interactions, it is concluded that the strengthening of hydrogen bonds in the past decade, a complete thermodynamic description of the self-association of many proteins and their interactions is concluded.
Abstract: Reviewing the thermodynamic parameters characterizing self-association and ligand binding of proteins at 25 OC, we find AGO, AHo, AS\", and ACpo are often all of negative sign. It is thus not possible to account for the stability of association complexes of proteins on the basis of hydrophobic interactions alone. We present a conceptual model of protein association consisting of two steps: the mutual penetration of hydration layers causing disordering of the solvent followed by further short-range interactions. The net AGO for the complete association process is primarily determined by the positive entropy change accompanying the first step and the negative enthalpy change of the second step. On the basis of the thermochemical behavior of small molecule interactions, we conclude that the strengthening of hydrogen bonds in the I n the past decade, a complete thermodynamic description of the self-association of many proteins and their interactions From the Laboratory of Molecular Biology (P.D.R.) and Laboratory of Nutrition and Endocrinology (S.S.), National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20205. Received September 23, 1980. low dielectric macromolecular interior and van der Waals' interactions introduced as a consequence of the hydrophobic effect are the most important factors contributing to the observed negative values of AHo and ASo and hence to the stability of protein association complexes. The X-ray crystallographic structures of these complexes are consonant with this analysis. The tendency for protein association reactions to become entropy dominated and/or entropy-enthalpy assisted at low temperatures and enthalpy dominated at high temperatures (a consequence of the typically negative values of AC,\") arises from the diminution of the hydrophobic effect with increasing temperature which is a general property of the solvent, water. with small molecular substrates has become available. Concomitantly, X-ray crystallography has provided a detailed picture of some of these associations, and this has stimulated a number of theoretical studies (Levitt & Warshel, 1975; Gelin & Karplus, 1975; Chothia & Janin, 1975), based upon energetic considerations, to account for these structures. The This article not subject to U S . Copyright. Published 1981 by the American Chemical Society T H E R M O D Y N A M I C S O F P R O T E I N A S S O C I A T I O N V O L . 2 0 , N O . 1 1 , 1 9 8 1 3097 Table I: Thermodynamics of Protein Association' association process A G \" ~ AiY A s o , A c p o (kcal mol-') (kcal mol-l) (cal K-I mol-') (cal K-I mol-I) refb trypsin (bovine) + inhibitor (soybean) -14.6 8.6 78 -440 c, d deoxyhemoglobin S gelation -3.4 2.0 18 -200 e, f lysozyme self-association (indefinite) -3.9 -6 .4 -8.3 g glucagon trimerization -12.1 -3 1 -64 -430 h, i hemoglobin t haptoglobin -11.5 -3 3 -7 3 -940 i a-chymotrypsin dimerization -7.1 -35 -9 5 k, I S-peptide + S-protein (ribonuclease) -13 -40 -90 -1100 m, n All thermodynamic parameters expressed per mole of complex formed except the indefinite association cases of hemoglobin S and lysozyme for which the mole refers to the monomeric protein reacted. Unitary entropy and free energy are given for processes of defined stoichiometry. Standard states are hypothetical 1 M protein, pH at which the reaction was measured. All pHs were close to 7 except for trypsin, pH 5, haptoglobin, pH 5.5, and glucagon, pH 10.5. All data for 25 \"C except glucagon, T = 30 \"C. ence is to calorimetric work and the second is to X-ray crystallographic structure determination. al. (1974). e Rosset al. (1977). Wishner e t al. (1975). g Banerjee et al. (1975). Johnson et al. (1979). * Sasaki et al. (1975). For each entry, the first referSweet et Baugh & Trowbridge (1972). Lavialle et al. (1974). Shiao & Sturtevant (1969). lVandlen &Tulinsky (1973). Hearn et al. (1971). Wyckoff e t al. (1970). methodology and problems involved in such calculations have been critically reviewed by NBmethy & Scheraga (1977). In this paper we review the thermodynamics of protein association processes for the examples best characterized in terms of their chemistry and structure. From this survey we find that the thermodynamic parameters AGO, Ai?, AS\", and ACpo are predominantly of negative sign. This result poses severe difficulties for interpretations of protein association based upon the entropically driven hydrophobic effect. The aim of this paper is to attempt to account for the signs and magnitudes of these thermodynamic parameters for protein association reactions in terms of known molecular forces and the thermochemistry of small molecule interactions.
3,960 citations
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TL;DR: In this article, a method for the rapid calculation of atomic charges in σ-bonded and nonconjugated π-systems is presented, where only the connectivities of the atoms are considered.
Abstract: A method is presented for the rapid calculation of atomic charges in σ-bonded and nonconjugated π-systems. Atoms are characterized by their orbital electronegativities. In the calculation only the connectivities of the atoms are considered. Thus only the topology of a molecule is of importance. Through an iterative procedure partial equalization of orbital electronegativity is obtained. Excellent correlations of the atomic charges with core electron binding energies and with acidity constants are observed. This establishes their value in predicting experimental data.
3,391 citations
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TL;DR: In this article, the fluorescence of various fluorophores by molecular oxygen has been studied in aqueous and nonaqueous solutions equilibrated with oxygen pressures up to 100 atm.
Abstract: Quenching of the fluorescence of various fluorophores by molecular oxygen has been studied in aqueous and nonaqueous solutions equilibrated with oxygen pressures up to 100 atm. Temperature dependence of quenching, agreement with the Stern–Volmer equation, and fluorescence lifetime measurements indicate that essentially all the observed quenching is dynamic and close to the diffusion-controlled limits. Studies of charged polyamino acids containing tryptophan show that oxygen quenching, in contrast to I−, is completely insensitive to charge effects. Ethidium bromide, when intercalated into double helical DNA, is quenched with 1/30th of the efficiency of the free dye in solution. Three dyes bound to bovine serum albumin were also found to be relatively protected from the free diffusion of oxygen. Quenching of intrinsic or bound fluorophores by molecular oxygen is therefore an appropriate method to determine the accessibility to oxygen of regions of the macromolecule surrounding the fluorophore and indirectly the structural fluctuations in the macromolecule that permit its diffusion to the fluorophore.
2,300 citations
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TL;DR: The results of the model compound study provide evidence for a mechanism that follows the classical Stern-Volmer law (1919), predominantly involving collisional quenching, and illustrate the importance of local charge and solvent viscosity.
Abstract: The effect of iodide on the tryptophyl fluorescence of model compounds and of lysozyme was studied in order to evaluate the factors that determine the use of iodide as a selective quencher of the fluorescence of tryptophyl side chains of proteins exposed to solvent. The results with the model compounds indicate the involvement of a collisional quenching mechanism due to the agreement with the Stern-Volmer law and the proportionality of the quenching constant with To7 for indole-3-acetamide. Bimolecular rate constants, k a , calculated from measured quenching constants using available lifetime data are equal to, greater than, or less than 4-6 X lo9 M-' sec-l for uncharged, positively charged, and negaI n a preliminary study it was shown that a large fraction of the tryptophyi fluorescence of lysozyme in aqueous solution was quenched by low concentrations of iodide ion (Lehrer, lY67). It was concluded from a study of the magnitude of the quenching of fluorescence and the character of the difference fluorescence spectrum produced in the presence and absence of substrate that the fluorescence of tryptophyls exposed to solvent and located in the substrate binding site was preferentially quenched by iodide. It appeared that this technique, which can be called solute perturbation of protein fluorescence, could be used as a probe of fluorophor exposure in proteins in a manner analogous to the technique of solvent perturbation of protein absorption (Herskovits and Laskowski, 1960; Laskowski, 1966). * From the Department of Muscle Research, Boston Biomedical Research Institute, Boston, Massachusetts 021 14, and from the Department of Neurology, Harvard Medical School, Boston, Massuchusetts 02115. Receired April 22, 1971. This work was supported by grants from the National Institutes of Health (AM 11677 and HE 0581 1) and the iMass'ichuserts Heart Association (516). tively charged tryptophyl compounds, respectively. A modified version of the Stern-Volmer law was calculated for a fluorophor population with different quantum yields and quenching constants. This formulation allows the calculation of the effective quenching constant from the intercept and the slope at low iodide concentration of a F o ] M cs. l/(I-) plot. Data obtained for lysozyme indicate that for the native protein about one-half the tryptophyl fluorescence is accessible at pH 5.3 whereas all of the tryptophyl fluorescence is accessible in 6 M G d n . HCI. Information regarding the presence of charged groups near tryptophyl side chains was obtained for lysozyme by studying the dependence of the quenching on pH. More recently, studies by other workers have ~ised bromate (Winkler, 1969) and iodide (Arrio er al., 1970) to quench extrinsic fluorescence (Teale and Badley, 1970). Oxygen has also been used as a quencher of pyrenebutyric acid bound to proteins (Vaughan and Weber, 1970). Burstein (1968a) has also independently studied the quenching of tryptophyl fluorescence in model compounds by iodide. In order to learn more about the quenching mechanism and the factors which determine fluorophor exposure, various tryptophyl model compounds and a model protein, lysozyme. were used in the present study. The results of the model compound study provide evidence for a mechanism that follows the classical Stern-Volmer law (1919), predominantly involving collisional quenching, and illustrate the importance of local charge and solvent viscosity. The quenching of lysozyme fluorescence by iodide also appears to follow a similar mechanism because of the agreement obtained with a inodified version of the Stern-Volmer law which was calculated for a heterogeneous distribution of fluorophors in a protein. Effective Stern-Volmer quenching constants and values for the fractional accessible fluorescence were obtained for lyso3254 B I O C H E M I S T R Y , V O L . 1 0 , N O . 1 7 , 1 9 7 1 I O D I D E Q U E N C H I N G O F P R O T E I N F L U O R E S C E N C E zyme in 6 M Gdn.HCI, 'S M urea, and in aqueous solution at different pH's using the modified Stern--Volmer law. Values obtained are consistent with information regarding accessibility obtained by other methods. Experimental Section Muteriais. The following high-purity compounds were used as obtained from Mann Research Laboratories, New York, N. Y. : indole-3-acetic acid, indole-3-propionic acid, indole-3-butyric acid, indole-3-acetamide, N-Ac-L-TrpNH?, L-TrpOEt, Gdn . HCI, and urea. L-Trp (Cyclo Chemical Corp., Los Angeles), KI , Na&03, citric acid, and NaCl (Fisher Scientific Co., Freehold, N. J.) were all of high purity and used as obtained. Indole (Fisher) and skatole (3-methylindole) (Mann) were recrystallized from methanol containing Norit A (Matheson Coleman & Bell, Rutherford, N. J.). Hepes buffer was used as obtained from Calbiochem (Los Angeles). Poly(Glug9Trp1) and poly(Lysg7Trp3) were high molecular weight random sequence copolymers kindly supplied by Dr. G. Fasman. Lysozyme from two different sources were used (twice crystallized from Worthington Biochemical Corp., Freehold, N. J., and six-times crystallized from Miles Laboratories, Elkhart, Ind.). Both preparations gave similar results. Ac3Glcn was kindly supplied by Dr. J. Rupley and glycol chitin was obtained from Miles Laboratories. Methods. Quenching measurements at constant pH were made on five solutions of a given material containing increasing amounts of K I (0-0.2 M). These were prepared by diluting stock solutions of the model compound, of KI, of NaC1, and of buffer, into volumetric flasks. NaCl was used to keep the ionic strength constant. Stock solutions of the indole compounds were used within a few days of preparation and kept in the dark at 0-5" overnight. A small amount of SO3?(ca. M) was added to the iodide solution to prevent 1 3 formation. This was necessary because Isabsorbs in the wavelength region of tryptophyl fluorescence (filter effect) and because of possible chemical reaction. The solutions were equilibrated at 25 O before the measurements. Stock solutions of lysozyme were routinely filtered through a Millipore filter (HAWP 0.45 p ) before use. pH titrations were performed in the absence and presence of iodide by adding small quantities of 0.5 M HC1 to the solution in the cuvet, which contained 2 mM Hepes and 2 mM citrate, originally pH 8, then measuring the pH and fluorescence. pH was measured with a Radiometer PHM4c meter standardized at pH 4 and 7. Fluorescence spectra and intensities were measured by exciting a t 280 nm or longer. In most cases no corrections for iodide absorption were necessary. The fluorescence of a reference (usually the 0.2 M NaC1-0.0 M K I solution) was measured just before measuring the fluorescence of each solution in order t o correct for any exciting lamp fluctuation. Fluorescence measurements were made with either an Aminco-Bowman spectrofluorometer or an instrument that employs two Jarrell-Ash 0.25-m monochromators, an EM1 9601 B photomultiplier, and either a high-pressure 200-W mercury lamp or a 150-W high-pressure xenon lamp. Low temperatures were obtained with a refrigerated water circulator attached to the sample housing. The temperature was measured by inserting a calibrated thermistor into the sample solution. Abbreviations used are: Gdn . HCI, guanidine hydrochloride; Trp, tryptophyl or tryptophan; Hepes, N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid ; Ac3GIcii. tri-N-acetyl-D-glucosamirie. The activity of lysozyme was determined by the method of Hamaguchi et a/ . (1960). The decrease in viscosity with time caused by hydrolysis of glycol chitin (2 mg/ml) by lysozyme (0.02 mg/ml) in the presence of 0.2 M NaCl or 0.2 M KI in 2 m M citrate (pH 5.5) is the basis of this method. The specific viscosity of glycol chitin solutions in Cannon viscometers at 25" was measured with time after a small volume of lysozyme was added. The slope of the approximately linear viscosity decrease between 1 and 10 min was used as a measure of activity. The optical rotatory dispersion and circular dichroism spectra of lysozyme (0.95 mg/ml) in 0.2 M NaCl or in 0.2 M KI , 2 mM citrate (pH 5.2) were measured in a 1-cm cell with a Jasco spectropolarimeter. The absorbance of Iprevented measurements below 265 nm. Difference spectra were either measured with a Cary 15 or a Beckman DK spectrophotometer using mixing cells (Pyrocell, Inc., N. Y . ) . The total absorption over the wavelengths scanned was always below 2.2. The low-temperature studies were performed with a Beckman D K using a refrigerated sample holder.
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