Interaction of chlorpromazine with myoglobin and hemoglobin: A comparative study
TL;DR: Thermodynamic analysis revealed that binding of CPZ to hemoglobin was exothermic, whereas binding to myoglobin was endothermic with a high entropic contribution, suggesting that CPZ binding toMyoglobin is hydrophobic in nature.
Abstract: The mode and nature of the binding of chlorpromazine (CPZ), a psychotropic drug, with myoglobin, a monomeric muscle protein, were studied spectrofluorometrically and the results compared with those from the binding of CPZ to hemoglobin, a tetrameric allosteric protein from red blood cells (RBC). CPZ interacted with myoglobin in a non-cooperative mode, with a binding constant of 8.4 × 103 M−1 in 0.145 M NaCl, pH 6.8, whereas in the case of hemoglobin this interaction was found to be positively cooperative with a binding constant of 4.2 × 103M−1. The interaction of CPZ with myoglobin was not influenced by the NaCl molarity of the solution, whereas CPZ interaction with hemoglobin significantly decreased with increasing NaCl molarity, indicating that CPZ-hemoglobin binding is mostly electrostatic in nature, whereas that of the CPZ-myoglobin complex is of a non-electrostatic type. Thermodynamic analysis revealed that binding of CPZ to hemoglobin was exothermic (ΔH° = − 2.65 kcal/mol), whereas binding to myoglobin was endothermic (ΔH° = + 1.39 kcal/mol) with a high entropic contribution (ΔS° = + 23 cal/degree/mol), suggesting that CPZ binding to myoglobin is hydrophobic in nature. Such contrasting binding features of this drug have been discussed in the light of a typical subunit interaction property present and absent in hemoglobin and myoglobin, respectively.
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TL;DR: Analysis of relevant spectroscopic data leads to the conclusions that two binding sites are involved in BSA–3HF interaction, and the interaction is slightly positively cooperative in nature with a similar binding constant.
Abstract: Recent studies have shown that various synthetic as well as therapeutically active naturally occurring flavonols possess novel luminescence properties that can potentially serve as highly sensitive monitors of their microenvironments in biologically relevant systems. We report a study on the interactions of bovine serum albumin (BSA) with the model flavonol 3-hydroxyflavone (3HF), using the excited-state proton-transfer (ESPT) luminescence of 3HF as a probe. Upon addition of BSA to the flavonoid solutions, we observe remarkable changes in the absorption, ESPT fluorescence emission and excitation profiles as well as anisotropy (r) values. Complexation of 3HF with protein results in a pronounced shift (20 nm) of the ESPT emission maximum of the probe (from lambda(max)(em) = 513 nm to lambda(max)(em) = 533 nm) accompanied by a significant increase in fluorescence intensity. The spectral data also suggest that, in addition to ESPT, the protein environment induces proton abstraction from 3HF leading to formation of anionic species in the ground state. Fairly high values of anisotropy are observed in the presence of BSA for the tautomer (r = 0.25) as well as anion (r = 0.35) species of 3HF, implying that both the species are located in motion-restricted environments of BSA molecules. Analysis of relevant spectroscopic data leads to the conclusions that two binding sites are involved in BSA-3HF interaction, and the interaction is slightly positively cooperative in nature with a similar binding constant of 1.1 - 1.3 x 10(5) M(-1) for both these sites. Proteins 2001;43:75-81.
157 citations
TL;DR: It is demonstrated that in the presence of H(2)O(2), HbA(1c) degrades DNA and protein more efficiently than Hb a(0) and formation of carbonyl content, an index of oxidative stress, is higher by H bA( 1c).
Abstract: HbA(1c), the major glycated hemoglobin increases proportionately with blood glucose concentration in diabetes mellitus. H(2)O(2) promotes more iron release from HbA(1c) than that from nonglycated hemoglobin, HbA(0). This free iron, acting as a Fenton reagent, might produce free radicals and degrade cell constituents. Here we demonstrate that in the presence of H(2)O(2), HbA(1c) degrades DNA and protein more efficiently than HbA(0). Formation of carbonyl content, an index of oxidative stress, is higher by HbA(1c). Compared to HbA(0), HbA(1c) is more rapidly autooxidized. Besides these functional changes, glycation also causes structural modifications of hemoglobin. This is demonstrated by reduced alpha-helix content, more surface accessible hydrophobic tryptophan residues, increased thermolability and weaker heme-globin linkage in HbA(1c) than in its nonglycated analog. The glycation-induced structural modification of hemoglobin may be associated with its functional modification leading to oxidative stress in diabetic patients.
107 citations
TL;DR: It is suggested that micro TOF-Q mass spectrometry is useful technique for drug binding studies after showing for the first time that BA isolated from a plant (Tephrosia calophylla) binds to HSA.
Abstract: Betulinic acid (BA) has anti cancer and anti-HIV activity and has been proved to be therapeutically effective against cancerous and HIV-infected cells. Human serum albumin (HSA) is the predominant protein in the blood. Most drugs that bind to HSA will be transported to other parts of the body. Using micro TOF-Q mass spectrometry, we have shown, for the first time that BA isolated from a plant (Tephrosia calophylla) binds to HSA. The binding constant of BA to HSA was calculated from fluorescence data and found to be KBA = 1.685 ± 0.01 × 106 M−1, indicating a strong binding affinity. The secondary structure of the HSA–BA complex was determined by circular dichroism. The results indicate that the HSA in this complex is partially unfolded. Further, binding of BA at nanomolar concentrations of BA to free HSA was detected using micro TOF-Q mass spectrometry. The study revealed a mass increase from 65199 Da (free HSA) to 65643 Da (HSA + drug), where the additional mass of 444 Da was due to bound BA. Based on the results of this study, it is suggested that micro TOF-Q mass spectrometry is useful technique for drug binding studies.
105 citations
TL;DR: Information relating to the toxic and beneficial effects of these drugs has been discussed in this review and appropriate clinical application of phenothiazines can be developed in the future after gaining a comprehensive knowledge about their function.
Abstract: The phenothiazine group of drugs has long been known as antipsychotic drugs and previously it was extensively used for the treatment of anxiety Several pieces of evidence have shown that they interfere with a variety of cellular processes and in vitro can interact with biomolecules like DNA, proteins etc Recent reports have also revealed some new properties like antimicrobial, antiprionic, anticancerous activities of certain members of the phenothiazine group Appropriate clinical application of phenothiazines can be developed in the future after gaining a comprehensive knowledge about their function Information relating to the toxic and beneficial effects of these drugs has been discussed in this review
93 citations
Cites background from "Interaction of chlorpromazine with ..."
...A single-site protein, such as myoglobin, cannot accomplish this sort of communication, as one myoglobin molecule is completely independent of the state of the other and thus the interaction of phenothiazines with myoglobin is found to be non-cooperative in nature (Bhattacharyya et al., 1994)....
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...Such interaction is found to be cooperative in nature (Bhattacharyya et al., 1990, 1994)....
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Abstract: The interaction between morin–Eu(III) complex and calf thymus DNA in physiological buffer (pH 7.4) was investigated using UV–vis spectrophotometry, fluorescence spectroscopy, viscosity measurements and DNA melting techniques. Hypochromicity and red shift of the absorption spectra of morin–Eu(III) complex were observed in the presence of DNA, and the fluorescence intensity of morin–Eu(III) complex was greatly enhanced with the addition of DNA. Moreover, fluorescence quenching and blue shift of the emission peak were seen in the DNA–ethidium bromide (EB) system when morin–Eu(III) complex was added. The relative viscosity of DNA increased with the addition of morin–Eu(III) complex, whereas the value of melting temperature of DNA–EB system decreased in the presence of morin–Eu(III) complex. All these results indicated that morin–Eu(III) complex can bind to DNA and the major binding mode is intercalative binding. The 3:1 morin:Eu(III) complex (estimated binding constant = 2.36 × 106 L mol−1) is stabilized by intercalation into the DNA. The calculated binding constants of morin–Eu(III) complex with DNA at 292, 301 and 310 K were 7.47 × 104, 8.89 × 104 and 1.13 × 105 L mol−1, respectively. The thermodynamic parameters were also obtained: ΔHθ was 20.14 kJ mol−1 > 0 and ΔSθ was 161.70 J mol−1 K−1 > 0, suggesting that hydrophobic force plays a major role in the binding of morin–Eu(III) complex to DNA.
82 citations
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518 citations
TL;DR: Binding of chlorpromazine with human hemoglobin has been studied by equilibrium dialysis and fluorescence quenching and results revealed that the binding was positively cooperative with overall affinity constant K = 3.8 x 10(3) M-1.
Abstract: Binding of chlorpromazine (CPZ) with human hemoglobin has been studied by equilibrium dialysis and fluorescence quenching. Results of equilibrium dialysis experiment when analysed by Hill plot revealed that the binding was positively cooperative with overall affinity constant K = 3.8 × 103 M−1. CPZ quenched the fluorescence of hemoglobin and the analysis of the quenching data by Stern-Volmer equation indicated two types of quenching process, namely, dynamic and static quenching. Dynamic quenching constant was measured from the decay of fluorescent life time of tryptophans of hemoglobin in presence of CPZ. Static quenching constant concerned with the ground state complex formation between CPZ and hemoglobin was found to be 5 × 103 M−1. Almost all the tryptophans of hemoglobin were found to be accessible for CPZ to interact with. Oxygen was found to be released when CPZ was added to hemoglobin. Extent of release of oxygen depends on the D P ratio of CPZ(D): hemoglobin(P).
99 citations
TL;DR: This chapter presents procedures for the isolation of intracellular oxygen-binding proteins of tissues, called “tissue hemoglobins” in the widest sense, which are monomers or dimers having a minimum molecular weight of 18,000 with similar optical spectra and chemical reactivity.
Abstract: Publisher Summary This chapter presents procedures for the isolation of intracellular oxygen-binding proteins of tissues, called “tissue hemoglobins” in the widest sense. All of these, except Ascaris and yeast hemoglobin, are monomers or dimers having a minimum molecular weight of 18,000 with similar optical spectra and chemical reactivity. Strictly, only muscle hemoglobin should be called “myoglobin”; by extension the term is often applied to other tissue hemoglobins as well. Ferric myoglobin may be purified by chromatography on carboxymethyl (CM) cellulose, usually at slightly acid pH or on diethylaminoethyl (DEAE) cellulose. The choice of preparative procedure depends on the use to which the purified myoglobin will be put. Both DEAE and CM ion-exchange columns yield myoglobin that is pure in the sense of being free from contaminating polypeptide chains. Better resolution of forms of myoglobin differing only in charge is achieved on CM-cellulose. Such columns, however, are usually operated at acid pH, and it is a matter of experience that oxymyoglobin exposed to mildly acidic conditions becomes ferric and, in the process, undergoes some minor but apparently irreversible change. The chapter also explains the isolation and purification of vertebrate myoglobins.
73 citations
TL;DR: The irreversible binding of chlorpromazine radical cation (CPZ+.) and photoactivated chlor Promazine ( CPZ) to calf thymus DNA in vitro and bacterial macromolecules in intact bacterium cells was investigated and the consequences of covalent binding for the cytotoxicity and genotoxicity of CPZ+.
Abstract: The irreversible binding of chlorpromazine radical cation (CPZ+·) and photoactivated chlorpromazine (CPZ) to calf thymus DNA in vitro and bacterial macromolecules in intact bacterium cells was investigated. CPZ+· may be formed in vivo metabolically and photochemically. CPZ+· and photoactivated CPZ bind covalently to double- and single-strand DNA. The conformation of the DNA appeared to be important for the CPZ+· reactivity: though CPZ+· is less stabilized by complex formation with single-strand DNA, the reaction rate and the binding capacity of DNA-complexed CPZ+· with single-strand DNA is larger than with double-strand DNA. Photoactivated CPZ binds considerably to proteins, DNA and RNA in the intact bacterium cells. In spite of the relatively short lifetime of CPZ+· in the presence of the cells CPZ+· also binds irreversibly to bacterial DNA, RNA and proteins. The consequences of covalent binding for the cytotoxicity and genotoxicity of CPZ+· and photoactivated CPZ and the possible role for CPZ+· as an intermediate in the photobinding of CPZ is discussed.
38 citations
TL;DR: Scatchard analysis indicates that all four ligands are true agonists of the receptor exhibiting positive cooperative binding with the existence of more than one class of binding site.
Abstract: Fluorimetry and spectrophotometry have been used to study the binding of dimethyl, dipropyl, dibutyl and diphenylnitrosamine to nicotinic acetylcholine receptor isolated, and purified, from Torpedo fuscomaculata . Scatchard analysis indicates that all four ligands are true agonists of the receptor exhibiting positive cooperative binding with the existence of more than one class of binding site. The number of binding sites for the nitrosamines approximates 2. Diphenylnitrosamine binds to the receptor more tightly at low concentrations (Kd1 = 1.3 μM) than the aliphatic nitrosamine (Kd1 = 8–12 μM). Yet at high concentrations all nitrosamines behaved with similar Kd values (27–38 μM).
29 citations