Molecular factors affecting the complex formation between deferiprone (L1) and Cu(II). Possible implications on efficacy and toxicity.

Deferiprone (1,2-dimethyl-3-hydroxypyrid-4-one, L1, CAS 30652-11-0) is a new chelating drug used worldwide for the treatment of iron overloading conditions. Spectrophotometric and potentiometric measurements were carried out to investigate the interaction of L1 with Cu(II) ions under different conditions. The complexation of Cu(II) ions with L1 in aqueous solution leads predominantly to the formation of the Cu(L1)2 species at a pH range of 4-9. The experimental results indicate that L1 has high affinity for Cu(II) with stability constants log beta 11 = 10.3 +/- 0.9 and log beta 12 = 19.2 +/- 0.6. The effect of Cu(II) ions on the affinity of L1 for Fe(III) ions by competition reactions in vitro indicate displacement of Fe(III) in a concentration dependent manner by Cu(II). Similarly, the presence of different buffers at various pH values resulted in the formation of different stoichiometry L1 complexes with Cu(II) and of mixed complexes with buffer anions. The strong interaction of L1 with Cu(II) may have implications on the therapeutic and toxicological properties of this chelating drug. In particular, L1 may be used in the treatment of copper overloading conditions, such as Wilson's disease or other conditions where copper toxicity is implicated.     

The influence of Cd²⁺, Hg²⁺ and Pb²⁺ on taxifolin binding to bovine serum albumin by spectroscopic methods: with the viewpoint of toxic ions/drug interference

The effect of heavy metal ions, Cd(2+), Hg(2+) and Pb(2+) on taxifolin binding to bovine serum albumin (BSA) has been investigated by spectroscopic methods. The results indicated that the presence of heavy metal ions significantly affected the binding modes and binding affinities of taxifolin to BSA, and the effects depended on the type of heavy metal ions. One binding mode was found for taxifolin with and without Cd(2+), while two binding modes-a weaker one at low concentration and a stronger one at high concentration-were found for taxifolin in the presence of Hg(2+) and Pb(2+). Cd(2+) decreased the binding affinity of taxifolin for BSA by 7.3%; however, Hg(2+) increased the binding affinity of taxifolin for BSA by 13.3% in lower concentration and 33.3% in higher concentration. Pb(2+) decreased the binding affinity of taxifolin for BSA by 28.4% in lower concentration, and increased the binding affinity of taxifolin for BSA by 20.6% in higher concentration. The decreased binding affinity of taxifolin for BSA in the presence of Cd(2+) was mainly because of the existence of competitive binding between taxifolin and Cd(2+). However, the conformational change of BSA may the main reason for the changed binding affinity and binding distance of taxifolin for BSA in the presence of Hg(2+) and Pb(2+).     

Analysis of binding interaction between puerarin and bovine serum albumin by multi-spectroscopic method

The interaction of puerarin and bovine serum albumin (BSA) was investigated by means of fluorescence spectroscopy, resonance light-scattering spectroscopy, infrared spectroscopy, and synchronous fluorescence spectra. The apparent binding constants (K(a)) between puerarin and BSA were 1.13 x 10(4) (20 degrees C), and 1.54 x 10(4) lmol(-1) (30 degrees C), and the binding sites values (n) were 0.95+/-0.02. The experimental results showed that the puerarin could be inserted into the BSA, quenching the inner fluorescence by forming the puerarin-BSA complex. The addition of increasing puerarin to BSA solution leads to the gradual decrease in RLS intensity, exhibiting the formation of the aggregate in solution. It was found that both static quenching and non-radiation energy transfer were the main reasons for the fluorescence quenching. The positive entropy change and enthalpy change indicated that the interaction of puerarin and BSA was driven mainly by hydrophobic forces. The process of binding was a spontaneous process in which Gibbs free energy change was negative. The competing binding reaction with BSA between Fe(3+), Cu(2+) and puerarin was investigated. The effect of Fe(3+) and Cu(2+) on the binding of puerarin with BSA is discussed.     

Protein–Peptide Interactions as Probed by Tryptophan Fluorescence: Serum Albumins and Enkephalin Metabolites

Binding of Leu-enkephalin and the enkephalin metabolite, tyrosineglycine-glycine (TGG), to bovine serum albumin (BSA) was studied as a model to investigate protein peptide interactions. TGG and Leu-enkephalin quench the tryptophyl fluorescence of BSA. Stern-Volmer quenching constants were typically in the range of 40 to 300 M^–1, depending on the experimental conditions. The addition of Cu(II) or Ni(II) did not change the quenching constant, indicating that TGG does not compete for the metal binding sites on BSA. From fluorescence quenching studies with TGG, tyrosyl-glycine, tyrosine and glycyl-glycine, it was concluded that the presence of the tyrosine residue is required for the observed quenching. The phenolic group in tyrosine accounted for the quenching mechanism because phenol was efficient in quenching BSA fluorescence, whereas phenylalanine had no detectable effect. A large solvent isotope effect on the quenching constant of phenol and TGG with BSA strongly suggests an active role of the –OH functionality in the quenching mechanism.     

Differences in binding properties of two proton pump inhibitors on the gastric H+,K+-ATPase in vivo

Restoration of acid secretion after treatment with covalently-bound proton pump inhibitors may depend on protein turnover and on reversal of inhibition by reducing agents such as glutathione. Glutathione incubation of the H(+),K(+)-ATPase isolated from omeprazole or pantoprazole-treated rats reversed 88% of the omeprazole inhibition but none of the pantoprazole inhibition. The present study was designed to measure binding properties of omeprazole or pantoprazole in vivo. Rats were injected with (14)C-omeprazole or (14)C-pantoprazole after acid stimulation. The specific binding to the gastric H(+),K(+)-ATPase was measured at timed intervals as well as reversal of binding by glutathione reduction. The stoichiometry of omeprazole and pantoprazole binding to the catalytic subunit of the H(+),K(+)-ATPase was 2 moles of inhibitor per mole of the H(+),K(+)-ATPase phosphoenzyme. Omeprazole bound to one cysteine between transmembrane segments 5/6 and one between 7/8, pantoprazole only to the two cysteines in the TM5/6 domain. Loss of drug from the pump was biphasic, the fast component accounted for 84% of omeprazole binding and 51% of pantoprazole binding. Similarly, only 16% of omeprazole binding but 40% of pantoprazole binding was not reversed by glutathione. The residence time of omeprazole and pantoprazole on the ATPase in vivo depends on the reversibility of binding. Binding of pantoprazole at cysteine 822 is irreversible whereas that of omeprazole at cysteine 813 and 892 is reversible both in vivo and in vitro. This is consistent with the luminal exposure of cysteine 813 and 892 and the intra-membranal location of cysteine 822 in the 3D structure of the H(+),K(+)-ATPase.     

Study of the interaction between mercury (II) and bovine serum albumin by spectroscopic methods

Mercury is a significant environmental pollutant that originates from industry. Mercury will bind with albumin and destroy biological functions in humans if it enters the blood. In this paper, the interaction between mercury (II) and bovine serum albumin (BSA) was investigated in vitro by fluorescence, UV–Vis absorption and circular dichroism (CD) under simulated physiological conditions. This study proves that the probable quenching mechanism of BSA by mercury (II) was mainly static quenching due to the formation of a mercury (II)–BSA complex. The quenching constant K_a and the corresponding thermodynamic parameters (ΔH, ΔS and ΔG) at four different temperatures were calculated by a modified Stern–Volmer equation and the van’t Hoff equation, respectively. The results revealed that the interaction between mercury (II) and BSA was mainly enthalpy-driven and that hydrogen bonding and van der Waals forces played a major role in the reaction. The obtained data for binding sites of n approximately equal to 1 indicated that there was a single class of binding site for the BSA with mercury (II). The value of the distance r (3.55 nm), determined by Föster's non-radioactive energy transfer theory, suggested that the energy transfer from BSA to mercury (II) occurred with a high probability. The conformational investigation from synchronous fluorescence, CD spectroscopy and three-dimensional fluorescence showed that the presence of mercury (II) resulted in micro-environmental and conformational changes of the BSA molecules, which may be responsible for the toxicity of mercury (II) in vivo.     

Antiradiation compounds: XXI. Metal ion complexation of bis(methylthio) and methylthio amino derivatives of 1-methylquinolinium-2-dithioacetic acids

With the assumption that the radiation-protective ability (whole body protection of mice) of the bis(methylthio) and methylthio amino derivatives of the 1-methylquinolinium-2-dithioacetic acids may be due to complexation of copper or zinc ions in vivo, the ability of these compounds to form stable metal complexes has been observed. Metal ion stability constants have been determined for three of the compounds with Al(III), Cu(II), Fe(III), and Zn(II) ions, and stabilities somewhat larger than those for simple peptides were found. Structures of the Cu(II) and Fe(III) complexes are proposed, based on elemental analyses and infrared absorption. The Cu(II) complexes are 1:1 ligand:metal structures that precipitated as double salts with CuCl2, and the Fe(III) complexes are 2:1 structures in which the ligands incorporated an additional sulfur atom.     

Site-specific DNA damage by phenylhydrazine and phenelzine in the presence of Cu(II) ion or Fe(III) complexes: roles of active oxygen species and carbon radicals.

Phenylhydrazine cleaved isolated DNA in the presence of Cu(II), Mn(III), hemin, Fe(III)-EDTA, or peroxidase/H2O2, while phenelzine cleaved in the presence of Cu(II). DNA cleavage by phenylhydrazine in the presence of Mn(III), hemin, or Fe(III)-EDTA occurred without marked site specificity. Inhibitory effects of scavengers of hydroxyl free radical (.OH) on the DNA damage suggest the involvement of .OH. On the other hand, Cu(II)-mediated DNA cleavage by phenylhydrazine or phenelzine was inhibited by catalase and bathocuproine, a Cu(I)-specific chelator, but not by .OH scavengers. The predominant cleavage site was the thymine residue of 5'-GTC-3' sequence. Since the cleavage pattern was similar to that induced by Cu(I) plus H2O2 but not to that induced by Cu(II) plus H2O2, it is speculated that the copper-oxygen complex derived from the reaction of H2O2 with Cu(I) participates in DNA damage by phenylhydrazine or phenelzine in the presence of Cu(II). A comparison between scavenger effects on the DNA damage and those on radical production detected with ESR suggests that carbon-centered radicals (phenyl radical, 2-phenylethyl radical) do not play an important role in Cu(II)-, hemin-, or Fe(III)-EDTA-mediated DNA damage by phenylhydrazine or phenelzine of relatively low concentrations (less than 0.5 mM). However, during the oxidation of a high concentration (10 mM) of phenylhydrazine by ferricyanide, phenyl radical seemed to cause DNA damage, especially the breakage of the deoxyribose phosphate backbone. The possibility that active oxygen species (copper-oxygen complex, .OH) are more important in DNA damage induced by hydrazines in vivo than carbon-centered radicals is discussed.     

亚甲蓝类似物的合成及其与牛血清白蛋白相互作用的研究

目的 合成3种亚甲蓝类似物3,7-二(二正丙胺基)-吩噻嗪-5-鎓碘化物、3,7-二(二正丁胺基)-吩噻嗪-5-鎓碘化物和3,7-二(二正戊胺基)-吩噻嗪-5-鎓碘化物,并研究模拟生理条件下这3种亚甲蓝类似物与牛血清白蛋白的相互作用.方法 通过1H-NMR及MS对这3种亚甲蓝类似物进行结构表征,应用荧光光谱法研究了模拟生理条件下这3种亚甲蓝类似物与牛血清白蛋白的相互作用.结果 3种亚甲蓝类似物和BSA相互作用形成了蛋白质-药物复合物并猝灭其固有荧光,亚甲蓝类似物与BSA相互作用过程的ΔG0<0,亚甲蓝类似物与BSA荧光残基间的距离r均小于7 nm.结论 亚甲蓝类似物和BSA相互作用的猝灭机制为静态猝灭,二者之间的反应是自发进行的.非辐射能量转移理论表明BSA与亚甲蓝类似物之间存在非辐射能量转移.同步荧光光谱和三维荧光光谱的研究结果表明,亚甲蓝类似物与BSA的相互作用导致BSA构象发生变化.     

Free radical production and site-specific DNA damage induced by hydralazine in the presence of metal ions or peroxidase/hydrogen peroxide.

Hydralazine caused site-specific DNA damage in the presence of Cu(II), Co(II), Fe(III), or peroxidase/H2O2. The order of inducing effect of metal ions on hydralazine-dependent DNA damage [Cu(II) greater than Co(II) greater than Fe(III)] was related to that of accelerating effect on the O2 consumption rate of hydralazine autoxidation. Catalase completely inhibited DNA damage by hydralazine plus Cu(II), but hydroxyl radical (.OH) scavengers and superoxide dismutase did not. On the other hand, DNA damage by hydralazine plus Fe(III) was inhibited by catalase and .OH scavengers. Hydralazine plus Cu(II) induced piperidine-labile sites predominantly at guanine and some adenine residues, whereas hydralazine plus Fe(III) caused cleavages at every nucleotide. Activation of hydralazine by peroxidase/H2O2 caused guanine-specific modification in DNA. ESR-spin trapping experiment showed that .OH and superoxide are generated during the Fe(III)- or Cu(II)-catalysed autoxidation of hydralazine, respectively, and that nitrogen-centered radical is generated during the Cu(II)- or peroxidase-catalysed oxidation. The generation of nitrogen-centered radical was also supported by HPLC-mass spectrometry. The results suggest that the guanine-specific modification by the enzymatic activation of hydralazine is due to the nitrogen-centered hydralazyl radical or derived active species, whereas .OH participates in DNA damage by hydralazine plus Fe(III). The mechanism of hydralazine plus Cu(II)-induced DNA damage is complex. The possible role of the DNA damage induced by hydralazine in the presence of Cu(II) or peroxidase/H2O2 is discussed in relation to hydralazine-induced lupus, mutation, and cancer.     

Study of the interaction of an anticancer drug with human and bovine serum albumin: spectroscopic approach

The interactions between gemcitabine hydrochloride (GEM) and bovine serum albumin (BSA) or human serum albumin (HSA) have been studied by spectroscopic techniques. By the analysis of fluorescence spectrum and fluorescence intensity, it was observed that the GEM has a strong ability to quench the intrinsic fluorescence of both BSA and HSA through a static quenching procedure. The association constants of GEM with BSA and HSA were determined at different temperatures based on fluorescence quenching results. The negative ΔH° and positive ΔS° values in case of GEM–BSA and GEM–HSA complexes showed that both hydrogen bonds and hydrophobic interactions play a role in the binding of GEM to BSA or HSA. Experimental results showed that the binding of GEM to BSA or HSA induced conformational changes in BSA and HSA. From the quantitative analysis data of CD spectra, the α-helix of 57.58% and 34.82% in free BSA and free HSA decreased to 40.82% and 29.84% in BSA–GEM and HSA–GEM complexes, respectively, and hence confirmed that the secondary structure of protein was altered by GEM. The interactions of BSA and HSA with GEM were also confirmed by UV absorption spectra. The distance, r, between donor (BSA or HSA) and acceptor (GEM) was obtained according to the Förster's theory of non-radiation energy transfer. The effects of common ions on the binding constants of both BSA–GEM and HSA–GEM complexes were also investigated.     

Basic aspects of selectivity of pantoprazole and its pharmacological actions

Pantoprazole sodium is a substituted benzimidazole derivative which controls acid secretion by inhibition of gastric H(+)/K(+)-ATPase. The prodrug pantoprazole accumulates in the acidic space of the parietal cell where it is converted to the pharmacologically active principle, a thiophilic cyclic sulfenamide. The pH-dependent activation profile, i.e., activation at pH 1 versus activation at pH 4-6, is more favorable for pantoprazole than for the other proton pump inhibitors (PPIs) currently available. In vitro, pantoprazole interferes less potently than omeprazole with biological targets not related to gastric acid secretion. The gastric target sites for the pantoprazole sulfenamide are the cysteines 813 and 822 of the catalytic subunit of the H(+)/K(+)-ATPase. In contrast to omeprazole, the two binding sites are located right at the proton channel. In rats, dogs and humans, pantoprazole produces marked and prolonged inhibition of both basal and stimulated acid secretion. Overall, its antisecretory potency is equal to that of omeprazole. Antiulcer activity has been demonstrated for pantoprazole in two rat models. As seen with H(2)-receptor antagonists and other PPIs, pantoprazole causes an increase in serum gastrin concentration which reflects the degree of gastric acid inhibition. Pantoprazole is mainly metabolized by CYP3A4 and 2C19, but displays a lower affinity for these phase I cytochrome P450 enzymes than omeprazole. In contrast to the latter, pantoprazole is further conjugated with sulfate by the hepatic phase II metabolism. These two differences may explain why pantoprazole does not interfere with the metabolism of any other drug thus far tested in humans.     

Study of the interaction between fluoroquinolones and bovine serum albumin

The mechanism of interaction between norfloxacin (NRF) and ciprofloxacin (CPF) with bovine serum albumin has been investigated using circular dichroism, fluorescence and absorption spectroscopy. The quenching mechanism of fluorescence of bovine serum albumin by fluoroquinolones was discussed. The binding sites number n and apparent binding constant K were measured by fluorescence quenching method. The thermodynamic parameters obtained from data at different temperatures were calculated. The distance r between donor (bovine serum albumin) and acceptor (fluoroquinolones) was obtained according to Forster theory of non-radiation energy transfer. The effect of common ions on binding constant was also investigated. The results of synchronous fluorescence spectra, UV–vis absorption spectra and circular dichroism of BSA in presence of fluoroquinolones show that the conformation of bovine serum albumin changed.     

In vitro study of the interaction between omeprazole and the metal ions Zn(II), Cu(II), and Co(II)

The purpose of this study was to investigate the possibility of a chemical interaction between omeprazole (OM) and the metal ions Zn(II), Cu(II), and Co(II). Using UV absorption spectroscopy and elemental analysis, it was demonstrated that all of the studied metals form complexes with OM. The spectral changes associated with the complexation reaction were used to obtain the stoichiometry and formation constants of the complexes. In all cases complexes were found to form in 1:2 metal to OM ratio. In the case of cobalt another complex species which appeared as a green precipitate was also evident. Copper was shown to form the complex with the formula Cu3(OM)2 in addition to Cu(OM)2. The complexation of cobalt and copper to OM was found to be time dependent and the time required for the completion of the reaction was determined (about 6 h). Apparent binding constants were also determined.     

Calorimetric and spectroscopic studies on the interaction of methimazole with bovine serum albumin

The potential binding interaction(s) of the anti-thyroid drug methimazole (MMZ) with the protein bovine serum albumin (BSA) has been studied using isothermal titration calorimetry (ITC) and UV-Visible, fluorescence and circular dichroism (CD) spectroscopic techniques. The binding of MMZ to BSA has been studied in both the presence and absence of added surfactants. Since, the ITC thermograms show the molar enthalpy of binding of MMZ and BSA to be zero within experimental error, either the enthalpy change of the binding interaction is zero or there is no binding occurring. The CD and the intrinsic fluorescence and life time spectra of BSA were unchanged by the addition of MMZ. This is also indicative of the absence of any significant interaction of MMZ with BSA.     

Interaction of triprolidine hydrochloride with serum albumins: thermodynamic and binding characteristics, and influence of site probes

The interaction between triprolidine hydrochloride (TRP) to serum albumins viz. bovine serum albumin (BSA) and human serum albumin (HSA) has been studied by spectroscopic methods. The experimental results revealed the static quenching mechanism in the interaction of TRP with protein. The number of binding sites close to unity for both TRP-BSA and TRP-HSA indicated the presence of single class of binding site for the drug in protein. The binding constant values of TRP-BSA and TRP-HSA were observed to be 4.75 ± 0.018 × 10(3) and 2.42 ± 0.024 × 10(4)M(-1) at 294 K, respectively. Thermodynamic parameters indicated that the hydrogen bond and van der Waals forces played the major role in the binding of TRP to proteins. The distance of separation between the serum albumin and TRP was obtained from the F?rster's theory of non-radioactive energy transfer. The metal ions viz., K(+), Ca(2+), Co(2+), Cu(2+), Ni(2+), Mn(2+) and Zn(2+) were found to influence the binding of the drug to protein. Displacement experiments indicated the binding of TRP to Sudlow's site I on both BSA and HSA. The CD, 3D fluorescence spectra and FT-IR spectral results revealed the changes in the secondary structure of protein upon interaction with TRP.     

(-)-Epigallocatechin gallate causes oxidative damage to isolated and cellular DNA

Green tea catechins, especially (-)-epigallocatechin gallate (EGCG), are believed to mediate much of the cancer chemopreventive effects of tea. However, it was reported that green tea catechins enhanced colon carcinogenesis in rats. Experiments using 32P-labeled DNA fragments obtained from human cancer-related genes showed that catechins induced DNA damage in the presence of metals such as Cu(II) and Fe(III) complexes. In the presence of Fe(III)EDTA, the order of DNA damaging ability was EGCG approximately (-)-epigallocatechin>(-)-epicatechin gallate>catechin. Catechins plus Fe(III)EDTA caused DNA damage at every nucleotide, most likely due to *OH generation from H(2)O(2). In the presence of Cu(II), the order was (-)-epigallocatechin>catechin>EGCG>(-)-epicatechin gallate. Cu(II)-mediated DNA damage by EGCG occurred most frequently at T and G residues, especially of 5'-TG-3' and GG sequences. Catalase and bathocuproine inhibited the Cu(II)-mediated DNA damage, suggesting the involvement of H(2)O(2) and Cu(I). In the presence of metal ions, increased amounts of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were found in DNA treated with EGCG. Furthermore, EGCG increased amounts of 8-oxodG in HL-60 cells, but not in the H(2)O(2)-resistant clone HP100. When GSH was reduced by L-buthionine-[S, R]-sulfoximine, a low concentration of EGCG increased amounts of 8-oxodG in HL-60 cells, further supporting the involvement of H(2)O(2) in cellular DNA damage. It is concluded that EGCG can induce H(2)O(2) generation and subsequent damage to isolated and cellular DNA, and that oxidative DNA damage may mediate the potential carcinogenicity of EGCG.     

黄芩素及黄芩苷与牛血清白蛋白结合作用比较研究及葡萄糖的影响

目的比较研究黄芩素和黄芩苷与牛血清白蛋白(BSA)分子间的结合作用及机制。方法通过光谱法比较研究黄芩素和黄芩苷与BSA结合作用,并观察葡萄糖对二者与BSA结合的影响。以能量传递原理和Lineweaver-Burk双倒数方程计算二者与BSA反应的结合常数和结合距离;以热力学参数判断二者与BSA间的作用力类型;以同步荧光技术考察黄芩素和黄芩苷对BSA构象的影响。结果黄芩素和黄芩苷与BSA反应的结合常数和结合距离均随着温度的升高而降低;与黄芩素相比,黄芩苷与BSA的结合距离增大,作用强度减弱。葡萄糖能明显增加二者与BSA的结合常数及结合位点。黄芩素与BSA的结合力为氢键和范德华力、黄芩苷为静电引力,从而导致BSA内在荧光静态猝灭。黄芩素和黄芩苷均能使BSA构象发生变化,黄芩素还能使BSA的色氨酸所处环境的疏水性降低。结论黄芩素分子上糖取代可降低其与BSA之间的结合作用并改变其作用力类型。生理浓度的葡萄糖可明显增加黄芩素和黄芩苷与BSA的结合常数及结合位点。     

金合欢素-7-O-葡萄糖苷与BSA相互作用的荧光光谱法研究

应用荧光光谱法研究了生理条件下金合欢素-7-O-葡萄糖苷与牛血清白蛋白（BSA）的相互作用。结果表明,其对BSA荧光的猝灭机制属于形成复合物的静态猝灭过程,并求得结合常数Ka为8.57×104L.mol-1,结合位点数n为1。根据热力学参数确定了其与BSA之间的主要作用力类型为静电作用力。采用同步荧光考察了药物对BSA构象的影响。此外,讨论了共存离子Cu2＋,Al3＋,Zn2＋,Mg2＋对药物与BSA结合作用的影响。