Human serum albumin interaction with paraquat studied using spectroscopic methods

The interaction between herbicide paraquat and human serum albumin (HSA) was investigated by fluorescence and UV/Vis absorption spectroscopy. Paraquat can strongly quench the intrinsic fluorescence of HSA by static quenching and nonradiative energy transferring; The hydrophobic and electrostatic interactions play a major role in stabilizing the complex. The binding site number n and apparent binding constant K_A, corresponding thermodynamic parameters ΔG, ΔH, ΔS at different temperatures, were calculated. The distance r between donor (HSA) and acceptor (paraquat) was obtained according to fluorescence resonance energy transfer. The effect of paraquat on the conformation of HSA was analyzed using synchronous fluorescence spectroscopy.     

环氧虫啶对苜蓿蚜的毒力及对其体内解毒酶活性的影响

新烟碱类杀虫剂环氧虫啶对非靶标生物具有潜在危害,但对其毒理机理、其在人体内的运输机制等方面缺乏研究。本研究运用荧光光谱法、分子对接、分子动力学、结合自由能计算和丙氨酸扫描等方法,研究了环氧虫啶与人血清白蛋白 (HSA) 的结合模式。结果表明：1) 环氧虫啶能有效猝灭HSA荧光,不同温度下环氧虫啶与HSA的结合常数在0.76 × 10⁵~1.57 × 10⁵ L/mol之间,具有较强的结合能力;2) 环氧虫啶结合在HSA的IIA疏水腔内,有1个结合位点;3) 结合自由能分析和热力学参数计算显示,环氧虫啶和HSA结合的主要作用力是范德华力和氢键作用;4) 分子动力学模拟结果显示,二者结合自由能为 ‒25.90 kJ/mol,形成了相对稳定的复合物;5) 丙氨酸突变扫描结果显示,氨基酸残基Gln459是环氧虫啶与HSA结合的关键氨基酸。该研究结果可为阐明环氧虫啶在人体内的毒性机理提供理论依据。

     

Studies on the interaction of aminocarb with calf thymus DNA by spectroscopic methods

The interaction between aminocarb and calf thymus DNA in physiological buffer (pH 7.4) was investigated by UV-vis spectrophotometry, fluorescence spectroscopy, viscosity measurements and DNA melting techniques. The absorption spectra of aminocarb with DNA showed a slight blue shift and hypochromic effect. Using ethidium bromide (EB) as a fluorescence probe, fluorescence quenching of the emission peak was seen in the DNA-EB system when aminocarb was added. The fluorescence polarization was gradually increased with increasing amounts of DNA. The value of melting temperature of DNA increased in the presence of aminocarb. Moreover, the relative viscosity of DNA increased with the addition of aminocarb. All the evidences indicated that the binding mode of aminocarb with DNA was an intercalative binding. The binding constants of aminocarb with DNA were determined. The calculated thermodynamic parameters suggested that the binding of aminocarb to DNA was driven mainly by hydrogen bond and van der Waals.     

Substrate binding by blasticidin S deaminase,an aminohydrolase for novel 4-aminopyrimidine nucleosides

The substrate specificity of blasticidin S deaminase (E.C. 3.5.4.23), purified from Aspergillus terreus, has been studied in detail. The enzyme was found to catalyze the hydrolytic deamination of cytosine nucleus in blasticidin S and its derivatives, while cytosine, cytidine, cytidine monophosphate, adenosine, and guanosine were not regarded as substrates. The structural requirements of compounds for binding by the enzyme were evaluated by the nature of inhibition and inhibitor constants; deaminohydroxyblasticidin S, the reaction product, and its derivatives inhibited the enzymatic aminohydrolysis of blasticidin S competitively. The K_i for deaminohydroxyblasticidin S was determined to be 2.3 × 10⁻⁵ M, which is close to the K_m for blasticidin S (2.1 × 10⁻⁵ M). The binding affinity of derivatives to the enzyme, −ΔG_bind, was calculated on the basis of K_i values, and further the partial binding affinities, −Δg, for several moieties or atomic groups in blasticidin S were deduced from the −ΔG_bind values. The results showed that the enzyme involves a specific binding site with multiple points corresponding to the carboxyl group of cytosinine moiety, the amide group, and the β-amino and guanidino groups of blastidic acid moiety in blasticidin S molecule; these facts are strongly indicative of the enzyme to be a new aminohydrolase for novel nucleosides such as blasticidin S.     

Binding of chlorpyrifos and cypermethrin to blood proteins

The features of two insecticides (chlorpyrifos and cypermethrin) binding to two blood proteins, bovine serum albumin (BSA), and bovine hemoglobin (BHb), were investigated via the fluorescence method. The results revealed that both insecticides caused the fluorescence quenching of BSA and the fluorescence enhancement of BHb. A new parameter (F_E), i.e., the fluorescence intensity when adequate insecticide was added, was introduced to obtain the association constant (K_A) and the number of binding sites (n). K_A and n of chlorpyrifos and cypermethrin binding to BSA were 2.99 × 10⁵ and 5.22 × 10⁵ L mol⁻¹, 1.25 and 0.78, respectively. K_A and n of chlorpyrifos and cypermethrin binding to BHb were 2.94 × 10⁴ and 2.48 × 10⁴ L mol⁻¹, 1.75 and 2.19, respectively. In conclusion, chlorpyrifos and cypermethrin could bind to BSA and BHb, and the binding of both insecticides to BSA was significantly stronger than that of insecticides to BHb. These could affect the distribution, metabolism, and excretion of insecticides.     

人血清白蛋白与氯氰菊酯相互作用的分子模拟研究

运用分子对接、分子动力学、结合自由能计算和丙氨酸扫描等分子模拟方法,研究了人血清白蛋白 (human serum albumin,HSA) 与氯氰菊酯的结合模式。结果表明:氯氰菊酯与HSA结合形成了稳定的复合物,与其氨基酸残基Arg209形成1个氢键,结合自由能为 –83.43 kJ/mol,其中范德华力是结合的主要驱动力,极性溶剂化能是主要抑制力。丙氨酸突变扫描结果显示,氨基酸残基Lys199的ΔΔG_bind值为16.78 kJ/mol,是HSA和氯氰菊酯结合的关键氨基酸。该研究结果为阐明氯氰菊酯在人体内的代谢机制提供了理论参考。     

Binding of phenthoate to bovine serum albumin and reduced inhibition on acetylcholinesterase

Organophosphorus pesticides (OPs) are of environmental significance due to their high toxicity to animals. Binding to plasma proteins may effective influence the toxicological properties of xenobiotics. In an attempt to evaluate the affinity of phenthoate (PTA) to bovine serum albumin (BSA) and inhibitory ability of bound PTA to acetylcholinesterase (AChE), we investigated the interactions between phenthoate (PTA) and bovine serum albumin (BSA) using tryptophan fluorescence quenching and subsequent inhibition on AChE activity by PTA. The results showed that PTA caused the fluorescence quenching of BSA because of the formation of a PTA-BSA complex. Quenching constants (K_sv), determined using the Sterns-Volmer equation to provide a measure of the binding affinity between PTA and BSA at 303, 306, 310 and 313 K were (3.4295 ± 0.0763) × 10⁻⁴, (3.2446 ± 0.0635) × 10⁻⁴, (3.0434 ± 0.0856) × 10⁻⁴ and (2.8262 ± 0.0569) × 10⁻⁴ M⁻¹, respectively. The thermodynamic parameters, ΔH and ΔS were −25.04 kJ mol⁻¹ and 168.94 J mol⁻¹ K⁻¹, respectively, which indicated that the electrostatic interactions played a major role in PTA-BSA association. The presence of BSA consistently reduced the inhibitory ability of PTA on AChE, with the relative activity being increased from 46.98 to 61.71% for the concentration range of BSA between 0 and 4.0 g L⁻¹.     

Interaction of spiro[(2R,3R,4S)-4-benzyloxy-2,3-iso-propylidenedioxy-1-oxacyclopentane-5,5′-(2-nitromethylene-1,3-diazacyclohexane)] with bovine serum albumin

The interaction of a novel pesticide, NMD (spiro[(2R,3R,4S)-4-benzyloxy-2,3-iso-propylidenedioxy-1-oxacyclopentane-5,5′-(2-nitromethylene-1,3-diazacyclohexane)]), with bovine serum albumin (BSA) has been investigated by using absorption, fluorescence, and circular dichroism (CD) spectroscopy methods. Quenching of the fluorescence of BSA has been observed in the presence of NMD. The binding parameters were determined using Stern–Volmer equation. From the thermodynamic parameters calculated according to the van’t Hoff equation, the enthalpy change ΔH, and entropy change ΔS were found to be −2.71 kJ mol⁻¹ and 82.56 J mol⁻¹ K⁻¹, respectively. These values suggested that, apart from an initial hydrophobic association, the complex is held together by van der Waals interactions and hydrogen bonding. These results provided a quantitative understanding of the binding of NMD to BSA, which is important in understanding its toxicity in vertebrates.     

Chemical modifications of the insecticide binding sites on human serum albumin: Role of indole,bilirubin, and fatty acid binding sites

Interaction of carbamate and organophosphorus insecticides with chemically modified human serum albumin was studied by solubilization and difference spectra of bound insecticides and quenching of albumin fluorescence. Chemical modification involved oxidation of 1 tryptophan and 4 histidine residues, acetylation of 4 tyrosine and 26 lysine residues, and reaction of 7 arginine residues with 1,2-cyclohexanedione. There was a decrease in binding due to modification of arginine, tryptophan, and tyrosine residues and an increased binding due to lysine acetylation. The presence of bilirubin and octanoate also decreased the binding of insecticides. Changes in fluorescence of bound ANS due to binding of insecticides to albumin indicate localized perturbation of albumin conformation. The binding of insecticides was not affected by palmitate at physiological concentrations (molar ratio less than 2) although higher fatty acid concentrations caused a decrease. These preliminary observations suggest that warfarin, indole, and bilirubin sites are probably among the multiple insecticide binding sites.     

荧光猝灭法测定梨小食心虫性诱剂含量以及田间应用

β-环糊精（β-CD）与中性红（NR）在碱性缓冲溶液中形成包合物（β-CD-NR）,使中性红的荧光强度F增大,而加入梨小食心虫性诱剂主要成分顺-8-十二碳烯醇乙酸酯（Z8-12：Ac）后,β-CD-NR的荧光发生猝灭,以此建立了测定Z8-12：Ac含量的新方法：在含有1.0 mL 1.0×10^-3 mol/L中性红、5.0 mL 1.0×10^-2 mol/L的β-CD、7.0 mL 0.2 mol/L的NaH₂PO₄-Na₂HPO₄缓冲溶液（pH 7.4）的荧光体系中,加入Z8-12：Ac,以双蒸水定容至25.0 mL,超声反应20 min后,将其放置在4℃的环境下冷却,得待测液。在激发波长为460 nm、发射波长为580 nm的条件下测定待测液的荧光强度F和β-CD-NR体系的荧光强度F₀,计算荧光猝灭值ΔF（ΔF＝F₀—F）。比较分析不同浓度的Z8-12：Ac标准溶液与相应的荧光猝灭值ΔF的关系,结果表明：在Z8-12：Ac浓度为1.0×10^-4～4.0×10^-4 mol/L范围内,荧光猝灭值ΔF与Z8-12：Ac的浓度c呈现良好的线性关系,线性方程为：ΔF＝28.3 c—16.8,R²＝0.9978。对2.0×10^-4 mol/L的Z8-12：Ac溶液进行11次平行测定,得出方法检出限为1.25×10^-5 mol/L（S/N＝3）,RSD为±1.5%,方法灵敏度和精密度良好。加标回收率在98.0%～103.0%,说明方法准确度良好。利用新方法测定了田间梨小诱芯中Z8-12：Ac随时间的残留量,统计了不同残留量对应的诱虫量。结果表明,本方法可以成功测定不同天数下诱芯中Z8-12：Ac的残留量,掌握了诱芯中Z8-12：Ac的衰减趋势,而且与实际诱虫量变化趋势基本吻合。本方法可为确保田间虫情监测预报的精确性以及保持性诱芯的诱杀效果提供技术支撑。     

Kinetic constants for the inhibition of acetylcholinesterase by phenyl carbamates

The kinetic constants of a variety of substituted phenyl N-methyl- and N,N-dimethylcarbamates, which inhibit bovine erythrocyte acetylcholinesterase, were determined by various experimental procedures. A procedure in the presence of a chromogenic substrate was developed, based on the suggestion of Hart and O'Brien, and was compared with the conventional Main method. The dissociation equilibrium constant, K_d, and the carbamylation rate constant, k₂, were shown to apparently depend on the inhibitor concentration range used for the determination in both procedures. Assuming that the binding of further inhibitor molecules to the reversible complex and the carbamylated enzyme is significant under conditions with high inhibitor concentrations, the concentration dependence of the kinetic constants was nicely delineated. It is indicated that reliable constants are obtainable with a rather low inhibitor concentration range, whose product by k_i is of the order of 0.2–1.0 min^?1.     

Comparative effects of two plant growth regulators; indole-3-acetic acid and chlorogenic acid on human and horse serum butyrylcholinesterase

Butyrylcholinesterase (BChE), a major detoxification enzyme found abundantly in many tissues and organisms, constitutes the first line defense in the serum of higher organisms and is a marker for toxic exposure. In this study, the interaction of two plant growth regulators, indole-3-acetic acid (IAA) and chlorogenic acid (CA) with purified human and horse serum BChE is investigated. The time dependent interaction of IAA with the two enzyme species was concentration dependent and rapid. Through kinetic studies, IAA was found to be linear-mixed type inhibitor for human serum BChE, and uncompetitive inhibitor for the horse serum enzyme. For the human BChE, α and the K_i value was calculated as 2.15 ± 1.09 and 3.09 ± 0.98 mM, respectively, and for the horse enzyme the K_i value was calculated as 1.05 ± 0.09 mM. The time dependent interaction of CA with the two enzyme species was biphasic. At low CA concentrations, CA stimulated the activities of both enzyme species whereas at high CA concentrations, inhibition was observed. At high concentrations, the inhibition kinetics for both enzymes fitted the non-competitive inhibition model. The K_i values calculated for human and horse BChE were 2.75 ± 0.14 and 0.96 ± 0.07 mM, respectively. The differences in the interaction of these two growth regulators with the two enzymes species arises from the structural differences between the human and horse serum BChE which can be considered as a triple human mutant BChE.     

Inhibition of P-glycoprotein ATPase and its transport function of Helicoverpa armigera by morin, quercetin and phloroglucinol

Flavonoids (morin, quercetin and phloroglucinol) were tested for their ability to modulate the function of P-glycoprotein ATPase of the insecticide resistant pest Helicoverpa armigera (Ha-Pgp). Flavonoids in the presence of ethylparaoxon or cypermethrin significantly reduced both larval weight as well as survival rate 40-50%. Morin and quercetin inhibited the activity of Ha-Pgp ATPase by 80-90%, whereas phloroglucinol inhibited ATPase activity by 40% at 100 μM concentration. These flavonoids inhibited the verapamil, ethylparaoxon and cypermethrin-stimulated Ha-Pgp ATPase activity. Morin, quercetin and phloroglucinol binding were quantitated by quenching of the intrinsic Trp fluorescence of purified Ha-Pgp ATPase. Drug transport was monitored in proteoliposomes containing Ha-Pgp ATPase using the high affinity fluorescent substrate tetramethylrosamine (TMR) in real time. Addition of the morin and quercetin mediated the collapse of the TMR concentration gradient generated by Ha-Pgp ATPase. The inhibition studies on Ha-Pgp ATPase activity may contribute towards understanding new strategies of the pest to overcome insecticide resistance.     

Structure-activity correlations of the insecticide Prolan and its analogs

Structure-activity correlations for 45 insecticidal diaryl nitropropanes (Prolan analogs) were analyzed by multiple regression analysis. Molecular bulk constants including van der Waal's radii, molar attraction constants, parachor, steric constants such as Taft's E₈ and Verloop's dimensional steric constants, hydrophobic constants such as II, and electronic parameters such as σ, F, and R were evaluated. It was concluded that the diaryl nitropropanes like the diaryl trichloroethanes fit into a receptor site which has an optimum volume for maximum interaction. The interaction between the insecticide and the receptor shows high correlation with steric constants for the aryl substituents and with intermolecular attractive forces. Highly asymmetrical compounds such as 1-(p-fluorophenyl)-1-(p-hexoxyphenyl)-2-nitropropane were surprisingly effective insecticides.     

Albumin and cytochrome P450 binding characteristics of juvenile hormone and its analogs

Binding data were gathered for the cecropia juvenile hormone (methyl(E, E cis)-10,11-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate) and two of its analogs {isopropyl(2E, 4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate; (E)-4-[(6,7-epoxy-3,7-dimethyl-2-nonenyl)-oxyl]-1,2-(methylenedioxy)benzene} with bovine serum albumin and rat hepatic microsomal cytochrome P₄₅₀. The proteins were found to bind the juvenile hormone and juvenile hormone analogs with affinity constants ranging from 10⁵ to 10⁶M^?1. Thermodynamic calculations suggest that the binding of all three compounds is electrostatic in nature and that the size of the ether and ester substituents can greatly influence the binding to proteins. The juvenile hormone and its analogs all formed spectrally apparent Type I complexes with oxidized cytochrome P₄₅₀; one of the juvenile hormone analogs formed a spectrally observable product adduct with reduced cytochrome P₄₅₀. The product complex may contribute many of the hormonal effects observed for this compound.     

In vitro inhibition of acetylcholinesterase by O,O-dimethyl S-aryl phosphorothioates

In vitro inhibition of house cricket head, house fly head, and bovine erythrocyte acetylcholinesterase by O,O-dimethyl S-aryl phosphorothioates was studied by Main's kinetic treatment. The potency of the compounds as reflected by the bimolecular reaction constants (k_i) indicated that house fly head acetylcholinesterase was the most sensitive to the inhibition followed by house cricket head and bovine erythrocyte acetylcholinesterase. There are no linear relationships between the phosphorylation rate constants and the total binding energies for the inhibition of three enzymes by this series of compounds, suggesting that the initial binding and the phosphorylation rate are not related. The structure and activity relationships were analyzed by multiple regression analyses with the use of Hammett's sigma, alkaline hydrolysis rates of the compounds, and p_i constants. The hydrophobic bonding of the compound on the enzyme surface as reflected by the p_i constant played a significant role in the determination of the potency of the inhibition of house cricket head and house fly head acetylcholinesterase by those compounds. However, the alkaline hydrolysis rates of the compounds, or the Hammett's sigma values seems to play a more important role in the determination of the inhibition of bovine erythrocyte acetylcholinesterase. Moderate insecticidal activity toward house crickets, house flies, and mosquito larvae were found.     

Effect of a nonhost-selective toxin from Alternaria alternata on chloroplast-electron transfer activity in Eupatorium adenophorum

AAC-toxin, a putative nonhost-selective phytotoxin, was obtained from Alternaria alternata causing a brown leaf spot disease of Crofton weed ( Eupatorium adenophorum ). The effect of AAC-toxin on the electron transfer reaction of chloroplasts showed that the activity of photosystem II, but not photosystem I, was completely inhibited by the toxin. AAC-toxin affected the following chlorophyll fluorescence parameters: coefficient of photochemical quenching ( q _P), the half-time value of fluorescence rise, and the O–J–I–P fluorescence induction kinetics curve, but not the ratio values of F _v/ F _m (the quantum yield of photosystem II) and the half-time value of fluorescence quenching. It was concluded that the toxin inhibited electron transfer from Q_A to Q_B (primary and secondary quinine acceptors of photosystem II) in photosystem II by competing with Q_B for the binding site in D1 protein on the thylakoid membrane.     

Properties of an antibody to Kelevan isolated by affinity chromatography: Antibody reactivation of ATPase activities inhibited by pesticides

Antibody molecules were produced by injection of BSA-Kelevan into chickens and rabbits. Pure antibody was obtained by a single pass of blood serum through an affinity column. The affinity gel was prepared by covalently binding BGG-Kelevan to activated Sepharose 4B-CN. Purity of the antibody was determined by ultracentrifugation and gel electrophoresis. Properties of the antibody included: sedimentation coefficient = 6.2, pI = 7.0, calculated MW = 150,000, and precipitin band formation using the microouchterlony test. The antibodies in free or immobilized form were able to prevent or reverse Kepone inhibition of ATPase activity from a variety of tissues from different sources. About 70 μg (approx 0.4 μM) of purified antibody was sufficient to restore the activity of mitochondrial (oligomycin-sensitive) Mg²⁺ ATPase activity which had been inhibited (in vitro) by 1 μM Kepone. The antibody was effective in preventing enzyme inhibition by other organochlorine pesticides with widely differing molecular structures. However, nonchlorinated inhibitors of mitochondrial oligomycin-sensitive Mg²⁺ ATPase activity were much less affected by the antibody. The available evidence suggests that the antibody binding site for the hapten may be specific for secondary or induced bonding forces due to the carbon-chlorine bonds rather than for a specific molecular structure.     

Cry1Ac的绿色荧光蛋白GFP标记及其在棉铃虫幼虫中肠的定位分布

为了明确Cry1Ac蛋白在棉铃虫体内与中肠组织的相互作用,采用重叠PCR方法将Bt-cry1Ac基因和绿色荧光蛋白GFP基因融合,构建含Cry1Ac毒蛋白和绿色荧光蛋白GFP原核表达载体,并在大肠杆菌大量表达。利用荧光显微镜观察发现,表达Cry1Ac-GFP融合蛋白的大肠杆菌在蓝光激发下发出绿色荧光。将含有融合蛋白的菌液拌入人工饲料饲喂3龄棉铃虫幼虫96h,取棉铃虫幼虫中肠做冰冻切片并在荧光显微镜下观察。结果显示,取食含有Cry1Ac-GFP融合蛋白饲料的棉铃虫幼虫中肠能够发出强烈荧光。比较Cry1Ac杀虫蛋白敏感和抗性棉铃虫幼虫中肠的发光部位,敏感棉铃虫幼虫的中肠围食膜已经消失,肠壁细胞发出强烈的荧光,而抗性棉铃虫的围食膜较健全并发出荧光。     

Effects of a new inhibitor K-23 on electron transport in photosystem II of higher plants

The effects of inhibitor K-23 on variable fluorescence, oxygen evolution and DCIP photoreduction were investigated. K-23 promotes the oxygen evolution and DCIP photoreduction at low concentration and inhibits them at relatively high concentrations, while an efficient inhibition at low concentration is found in variable fluorescence. These data further confirm that the inhibitor K-23 action is based on its redox interaction rather than quenching effect. Addition of DPC could not restore the DCIP photoreduction activity. It is suggested that the inhibitory site is at the acceptor side. Using ferricyanide as electron acceptor, the effect of K-23 and DCMU on the oxygen evolution of trypsin-treated thylakoids was investigated. It is found that oxygen evolution of trypsin-treated thylakoids was insensitive to DCMU, whereas became more sensitive to K-23 and also the promotion of K-23 at low concentration disappeared. This strongly indicates that trypsin treatment modified the binding site of K-23 and increased its accessibility to K-23 target site. From the comparison of K-23 with DCMU, we conclude that the binding site of K-23 is different from that of DCMU even though they both bind at the acceptor side.