Spectroscopic studies on interaction and sonodynamic damage of metallochlorophyllin (Chl-M (M=Fe, Zn and Cu)) to protein under ultrasonic irradiation

In this paper, the chlorophyll derivatives, metallochlorophyllin (Chl-M) (M=Fe, Zn and Cu) including chlorophyllin iron (Chl-Fe), chlorophyllin zinc (Chl-Zn) and chlorophyllin copper (Chl-Cu), were adopted as sonosensitizers to combine with ultrasonic irradiation, and the sonodynamic damage of bovine serum albumin (BSA) was investigated. At first, the interaction of Chl-M with BSA was studied by fluorescence spectroscopy. The results show that the quenching mechanism belongs to a static process and among them the affinity of Chl-Fe to BSA is the most obvious. Then, some influence factors on the sonodynamic damage of BSA molecules in the presence of Chl-M under ultrasonic irradiation were also studied. Synchronous fluorescence spectra show that the binding and damage sites of Chl-M to BSA molecule are mainly on the tryptophan (Trp) residues. The generation of ROS in Chl-M sonodynamic process is estimated by the method of Oxidation-Extraction Spectrometry (OEP). This paper may offer some valuable references for the study of the sonodynamic activity of Chl-M and the effect of the central metals. Synchronously, it contributes to the application of Chl-M in SDT for tumor treatment.     

Oxidation-extraction spectrometry of reactive oxygen species (ROS) generated by chlorophyllin magnesium (Chl-Mg) under ultrasonic irradiation

In order to examine the mechanism and process of sonodynamic reaction, the chlorophyllin magnesium (Chl-Mg) acting as a sonosensitizer was irradiated by ultrasound, and the generation of reactive oxygen species (ROS) were detected by the method of oxidation-extraction spectrometry (OES). That is, under ultrasonic irradiation in the presence of Chl-Mg, the 1,5-diphenyl carbazide (DPCI) is oxidized by generated ROS into 1,5-diphenyl carbazone (DPCO), which can be extracted by mixed organic solvent and display a obvious visible absorption at 563 nm wavelength. Besides, the generation conditions of ROS were also reviewed. The results demonstrated that the quantities of generated ROS increased with the increase of ultrasonic irradiation time, Chl-Mg concentration and DPCI concentration. Finally, several radical scavengers (l-Histidine (His), 2,6-Di-tert-butyl-methylphenol (BHT) and Vitamin C (VC)) were used to determine the kind of the generated ROS. It was found that at least the hydroxyl radical (OH) and singlet oxygen (1O2) were generated in the presence of Chl-Mg under ultrasonic irradiation. It is wish that this paper might offer some valuable references for the study on the mechanism of SDT and the application of Chl-Mg in tumor treatment.     

低频超声波照射下卟啉锰(HP-Mn)配合物对牛血清白蛋白(BSA)损伤的研究

应用紫外-可见(UV-vis)光谱和荧光光谱研究了卟啉-锰(HP-Mn)配合物与牛血清白蛋白(BSA)的相互作用及在超声波作用下HP-Mn对BSA的损伤作用,并探讨了超声波照射时间,HP-Mn浓度,酸度,离子种类和强度等因素对BSA损伤的影响。结果表明,HP-Mn对BSA荧光的猝灭属于静态猝灭,两者主要通过静电作用相互结合,同时也存在着配位作用,作用距离(r)为3.49 nm。另外,在超声波作用下,HP-Mn能够明显损伤BSA。损伤程度随着超声波照射时间的增长,酸度的升高和HP-Mn浓度的增大而增大,但离子种类和强度的影响较为复杂。这一结果为声动力学(SDT)疗法治疗肿瘤应用于临床具有重要的意义。     

Spectroscopic investigation on protein damage by ciprofloxacin under ultrasonic irradiation

In recent years, sonodynamic activities of many drugs have attracted more and more attention of researchers. The correlative study will promote the development of sonodynamic therapy (SDT) in anti-tumor treatment. In this work, bovine serum albumin (BSA) was used as a protein model to investigate the intensifying effects of ciprofloxacin (CPFX) ultrasonically induced protein damage by UV-vis and fluorescence spectra. Meanwhile, the conformation of BSA is changed upon the addition of CPFX and metal ions under ultrasound (US) so that the damaging site of BSA is considered. Various influencing factors, such as US irradiation time, metal ions, solution temperature and ionic strength, on the ultrasonically induced BSA damage are discussed. It was showed that CPFX could enhance ultrasonically induced BSA damage. The damage degree of BSA was aggravated with the increasing of US irradiation time, solution temperature, ionic strength as well as the addition of metal ions. Furthermore, the reactive oxygen species (ROS) in reaction system were detected by oxidation-extraction photometry (OEP). Experimental results also showed that US could activate CPFX to produce ROS, which were mainly determined as superoxide radical anion (.O2-) and hydroxyl radical (.OH).     

Spectroscopic study on interaction of bovine serum albumin with sodium magnesium chlorophyllin and its sonodynamic damage under ultrasonic irradiation

Sonodynamic therapy (SDT) is an attractive antitumor treatment for recent years. In this paper, sodium magnesium chlorophyllin (SMC) as a sonosensitizer combining with ultrasonic (US) irradiation to damage bovine serum albumin (BSA) has been investigated by fluorescence and UV–vis spectroscopy. The interaction of BSA with SMC was studied by the quenching of intrinsic fluorescence at varying temperature. The quenching constants (K_SV), effective binding constants (K_A), apparent association constants (K_a) and binding site numbers were determined. The results indicated the quenching mechanism is a static procedure. Thermodynamic parameters show that the interactions involve hydrogen bonds, hydrophobic interactions, electrostatic interactions and complexations. The binding distance is 3.533 nm. The synergistic effect of SMC and ultrasound was estimated including the study of damage conditions. Synchronous fluorescence spectra indicate the damage to Trp residues is more serious. This paper may offer some valuable references for using spectroscopy method to study the application of chlorophyll derivatives in antitumor treatment.     

Assisted sonodynamic damage of bovine serum albumin by metronidazole under ultrasonic irradiation combined with photosensitive antitumor drug-Amsacrine

By research, it was found that the Amsacrine (AMSA) can interact with bovine serum albumin (BSA). In this work, the AMSA was adopted as a sonosensitizer and the Metronidazole (MET) was used as a sensitizer to further damage BSA molecules under ultrasonic irradiation. It could be concluded that the damage degree of BSA molecules in the presence of AMSA and MET was more serious than in the presence of pure AMSA. That is, MET could aggravate the damage to BSA molecules under ultrasonic irradiation combined with AMSA. Meanwhile, the damage degree of BSA molecules was also influenced by some factors, such as ultrasonic irradiation time, MET concentration and solution acidity. In addition, the damage site of BSA molecules was estimated by synchronous fluorescence spectra. It was found that the tyrosine (Tyr) and tryptophan (Typ) residues were damaged almost averagely. Perhaps, these research results are of great significance for driving sonodynamic method to treat tumor in clinic application.     

Spectroscopic analyses on interaction of o-Vanillin-D-Phenylalanine, o-Vanillin-L-Tyrosine and o-Vanillin-L-Levodopa Schiff Bases with bovine serum albumin (BSA)

In this work, three o-Vanillin Schiff Bases (o-VSB: o-Vanillin-D-Phenylalanine (o-VDP), o-Vanillin-L-Tyrosine (o-VLT) and o-Vanillin-L-Levodopa (o-VLL)) with alanine constituent were synthesized by direct reflux method in ethanol solution, and then were used to study the interaction to bovine serum albumin (BSA) molecules by fluorescence spectroscopy. Based on the fluorescence quenching calculation, the bimolecular quenching constant (K(q)), apparent quenching constant (K(sv)), effective binding constant (K(A)) and corresponding dissociation constant (K(D)) as well as binding site number (n) were obtained. In addition, the binding distance (r) was also calculated according to Foster's non-radioactive energy transfer theory. The results show that these three o-VSB can efficiently bind to BSA molecules, but the binding array order is o-VDP-BSA>o-VLT-BSA>o-VLL-BSA. Synchronous fluorescence spectroscopy indicates that the o-VDP is more accessibility to tryptophan (Trp) residues of BSA molecules than to tyrosine (Tyr) residues. Nevertheless, the o-VLT and o-VLL are more accessibility to Tyr residues than to Trp residues.     

Spectroscopic Analysis of the Interactions of Anthraquinone Derivatives (Alizarin,Alizarin-DA and Alizarin-DA-Fe) with Bovine Serum Albumin (BSA)

In this paper, three anthraquinones (alizarin (1,2-dihydroxy-9,10-anthraquinone), alizarin-DA (1,2-dihydroxy-9,10-anthraquinone-3-aminomethyl-N,N-diacetic acid) and alizarin-DA-Fe (1,2-dihydroxy-9,10-anthraquinone-3-aminomethyl-N,N-diacetate-ferric(III))) with a tricyclic anthraquinone planar structure are used as quenchers, to study their interaction with bovine serum albumin (BSA) molecules by fluorescence spectroscopy. The results show that these three anthraquinones can bind to BSA molecules efficiently but the stabilities decrease in the order alizarin, alizarin-DA and alizarin-DA-Fe. In addition, synchronous fluorescence spectroscopy indicates that the tryptophan (Trp) residues of BSA molecules are more accessible to alizarin and alizarin-DA than the tyrosine (Tyr) residues, but both have similar accessibility to alizarin-DA-Fe.     

Spectroscopic analyses on interaction of Amantadine-Salicylaldehyde, Amantadine-5-Chloro-Salicylaldehyde and Amantadine-o-Vanillin Schiff-Bases with bovine serum albumin (BSA)

In this work, three Tricyclo [3.3.1.1(3,7)] decane-1-amine (Amantadine) Schiff-Bases, Amantadine-Salicylaldehyde (AS), Amantadine-5-Chloro-Salicylaldehyde (AS-5-C) and Amantadine-o-Vanillin (AS-o-V), were synthesized by direct heating reflux method in ethanol solution and characterized by infrared spectrum and elementary analysis. Fluorescence quenching was used to study the interaction of these Amantadine Schiff-Bases (AS, AS-5-C and AS-o-V) with bovine serum albumin (BSA). According to fluorescence quenching calculations the bimolecular quenching constant (K(q)), apparent quenching constant (K(SV)), effective binding constant (K(A)) and corresponding dissociation constant (K(D)), binding site number (n) and binding distance (r) were obtained. The results show that these Amantadine Schiff-Bases can obviously bind to BSA molecules and the binding strength order is AS相似文献   

Study on the sonodynamic activity and mechanism of promethazine hydrochloride by multi-spectroscopic techniques

In this paper, the bovine serum albumin (BSA) was selected as a target molecule, the sonodynamic damage to protein in the presence of promethazine hydrochloride (PMT) and its mechanism were studied by the means of absorption, fluorescence and circular dichroism (CD) spectra. The results of hyperchromic effect of absorption spectra and quenching of intrinsic fluorescence spectra indicate that the ultrasound-induced BSA molecules damage is enhanced by PMT. The damage degree of BSA molecules increases with the increase of ultrasonic irradiation time and PMT concentration. The results of synchronous fluorescence, three-dimensional fluorescence and CD spectra confirmed that the synergistic effects of ultrasound and PMT induced the damage of BSA molecules. The results of oxidation-extraction photometry with several reactive oxygen species (ROS) scavengers indicate that the damage of BSA molecules could be mainly due to the generation of ROS and both (1)O(2) and OH are the important mediators of the ultrasound-induced BSA molecules damage in the presence of PMT.     

Study on interaction between a new fluorescent probe 2-methylbenzo[b][1,10]phenanthrolin-7(12H)-one and BSA

A new fluorescence reagent, 2-methylbenzo[b][1,10]phenanthrolin-7(12H)-one (mBPO), synthesized in our laboratory was used as the probe for protein and its interaction with Bovine Serum Albumin (BSA) was investigated in detail in this paper. It was found that BSA had the ability to quench the fluorescence of mBPO at 411 nm (λ(ex) = 286 nm), and the quenched intensity of fluorescence was proportional to the concentration of BSA. Based on this fact, mBPO has been used as a fluorescence probe for the detection of BSA. Under the optimal conditions, the calibration graph is linear up to 0.5 mg L(-1) for BSA and the limit of detection (LOD) was 0.06 mg L(-1). The regression equation is y = 1048.8x + 7.2093 with R(2) = 0.9913. The mechanism for the interaction of mBPO with BSA was also studied, while the binding constant and the number of binding sites were calculated. According to the thermodynamics parameter, the binding mode between mBPO and BSA was deduced. The results suggested the interaction between mBPO and BSA to be hydrophobic force in nature. It also proved that the fluorescence quenching reaction was affected by the tryptophan residue of BSA. For there are two tryptophan (Trp) residues, in site 134 and site 212 of BSA, and mBPO maybe has interaction with them respectively.     

Catalytic damage of bovine serum albumin by metronidazole under ultrasonic irradiation in the presence of nano-sized TiO2 powder

In previous work, it was found that the bovine serum albumin (BSA) could obviously be damaged by nano-sized TiO₂ powder as a sonocatalyst under ultrasonic irradiation. In this work, metronidazole (MTZ) was adopted as a sensitizer to intensify the damage of BSA molecules. It was found that the damage degree of BSA molecules in the presence of MTZ was more serious than in the absence of MTZ. That is, under ultrasonic irradiation combined with nano-sized TiO₂ powder, the addition of MTZ could remarkably aggravate the damage to BSA molecules. Meanwhile, the damage degree was also affected by some influence factors, such as ultrasonic irradiation time, ultrasonic irradiation power, MTZ concentration, solution acidity, ionic strength and solution temperature. In addition, the damage site of BSA molecules was also estimated by synchronous fluorescence spectra. It was found that the damage site of BSA molecules was mainly at tyrosine (Tyr) residue.     

槲皮素与酪蛋白和牛血清白蛋白的相互作用及共存碳纳米管的影响

采用荧光猝灭和同步荧光法,研究了磷酸缓冲溶液(PBS, pH=7.4)中有无碳纳米管(CNTs)共存时,荧光活性物质槲皮素(Qct)与牛血清白蛋白(BSA)和酪蛋白(Cas)的相互作用. 推导了方法1(固定蛋白质浓度, 改变Qct浓度, 测量蛋白质荧光改变)和方法2(固定Qct浓度, 改变蛋白质浓度, 测量Qct荧光改变)研究分子间作用的一般方程, 由非线性最小二乘拟合法测算了结合常数K和摩尔结合比n, 并藉此定量评估了“光内滤所致猝灭”效应的影响. 研究了共存CNTs或Qct对BSA或Cas的荧光猝灭效应, 及CNTs对Qct-BSA和Qct-Cas相互作用的影响. 以同步荧光法考察了CNTs或Qct对BSA或Cas构象的影响, 并测算了CNTs或Qct与蛋白质中酪氨酸(Tyr)或色氨酸(Trp)残基相关的K和n. 结果表明, CNTs主要与处于蛋白质分子表面附近的Trp残基作用, 而小分子Qct则还可与处于蛋白质分子内部的Tyr残基作用.     

Interactions of Quercetin with Casein and Bovine Serum Albumin as well as the Effects of Coexisting Carbon Nanotubes

Fluorescence quenching and synchronous fluorescence methods were used to study the interactions of fluore-scence-active quercetin (Qct) with casein (Cas) and bovine serum albumin (BSA) in phosphate buffer solution (PBS, pH=7.4) with or without coexisting carbon nanotubes (CNTs). Formulae for binding constant (K) and molar binding ratio (n) were established for methods 1 (fixing protein concentration, changing Qct concentration, and monitoring the fluorescence of protein) and 2 (fixing Qct concentration, changing protein concentration, and monitoring the fluorescence of Qct), to which values of K and n were calculated via nonlinear least-square fitting of the experimental data, and the “optical inner filtering induced fluorescence quenching” effect was thus quantitatively evaluated. The quenching effects of coexisting CNTs on the fluorescence of Qct, BSA, and Cas, as well as the effects of coexisting CNTs on Qct-BSA and Qct-Cas interactions, were examined. Synchronous fluorescence was also used to examine the effects of coexisting CNTs and Qct on the conformations of BSA and Cas, with relevant K and n values for tyrosine (Tyr) and tryptophan (Trp) residues estimated. It was concluded that the CNTs mainly interacted with the Trp residues locating near the protein surfaces, but small-sized Qct molecules could further interact with the Tyr residues locating inside the protein molecules.     

Sonocatalytic damage of solute bovine serum albumin by disperse ZnO/porcine dens composite under ultrasonic irradiation

ZnO/Dens composite were prepared with nano-sized ZnO and porcine dens powders by mechanical mixing, liquid boiling, ultrasonic dispersion and heat-treated at 500°C for 50 min. The samples were characterized by X-ray diffraction (XRD) and its sonocatalytic activity was evaluated by the damage of bovine serum albumin (BSA). Furthermore, the effects of ultrasonic irradiation time, ZnO/Dens composite amount, solution acidity and ionic strength on the sonocatalytic damage of BSA were evaluated. The results show that the damage degree of BSA aggravated with the increase of ultrasonic irradiation time, ZnO/Dens composite amount and ionic strength, but weakened with the increase of solution acidity. In addition, the damage site to BSA molecules was analyzed by synchronous fluorescence spectra and the results exhibited that the damage site was mainly at tryptophan (Trp) residue. This study should be helpful to drive sonocatalytic method to treat tumors in clinic application.     

Interaction between bovine serum albumin and Indo-1 using fluorescence spectroscopic method

This work attempts to calculate the binding-site number using fluorescence spectroscopic method with bovine serum albumin (BSA) and Indo-1 as protein and ligand models, respectively. The method for calculating the binding-site number in BSA for Indo-1 was developed based on the relationships between changes in Indo-1 fluorescence intensity and the analytical concentration of BSA. The interaction between BSA with Indo-1 was investigated comprehensively using fluorescence techniques as well as fluorescence resonance energy transfer, and the thermodynamic parameters were calculated according to the effect of enthalpy on temperature. Three binding sites in BSA for Indo-1 were revealed, and the distances from Trp212 in BSA to the three binding sites were 2.93, 2.57 and 2.40 nm, respectively. It was also proven that Indo-1 embedded into the three hydrophobic cavities of BSA by hydrophobic association. This paper provides a reference on calculating the binding-site number in proteins for ligands and studying their interactions by fluorescence spectroscopic methods. In fluorescent quenching experiments, fluorescence changes were automatically recorded in real time by combining the Microlab 500 Series Dispenser and PTI fluorescence apparatus. __________ Translated from Chemical Journal of Chinese Universities, 2007, 28(2): 227–233 [译自: 高等学校化学学报]     

Ferroptosis promotes sonodynamic therapy: a platinum(ii)–indocyanine sonosensitizer

Sonodynamic therapy (SDT) has unique advantages in deep tumour ablation due to its deep penetration depth, showing great preclinical and clinical potential. Herein, a platinum(ii)–cyanine complex has been designed to investigate its potential as a SDT anticancer agent. It generates singlet oxygen (¹O₂) under ultrasound (US) irradiation or light irradiation, and exhibits US-cytotoxicity in breast cancer 4T1 cells but with negligible dark-cytotoxicity. Mechanistic investigations reveal that Pt-Cy reduces the cellular GSH and GPX4, and triggers cancer cell ferroptosis under US irradiation. The metabolomics analysis illustrates that Pt-Cy upon US treatment significantly dysregulates glutathione metabolism, and finally induces ferroptosis. In vivo studies further demonstrate that Pt-Cy inhibits tumor growth under US irradiation and its efficiency for SDT is better than that for PDT in vivo. This is the first example of platinum(ii) complexes for sonodynamic therapy. This work extends the biological applications of metal complexes from PDT to SDT.

A novel platinum(ii)–cyanine complex showed a greater excellent sonodynamic therapeutic effect than photodynamic therapy in vivo. This work expands the biological applications of metal complexes from traditional photodynamic therapy to sonodynamic therapy.     

Interaction of albumin with polysaccharides containing ionic groups

It is very important to clarify the mechanisms of the interaction between components of organisms. In this report, the interaction between bovine serum albumin (BSA) and ionic polysaccharides were discussed. The fluorescence spectrum of tryptophan (Trp) in BSA changed as its conformation changed. On adding polysaccharide containing sulfonic acid residues (sulfonic‐type) at pH 7.4, a remarkable blue shift of the emission maximum (λ_em) of Trp was observed. This blue shift was decreased by adding NaCl. In contrast, polysaccharide containing carboxylic acid residues (carboxy‐type) scarcely changed Trp fluorescence at the same pH. At a pH lower than the isoelectric point (PI = 4.7–4.9) of BSA, a remarkable blue shift was observed not only by adding sulfonic‐type polysaccharide but also by adding carboxy‐type polysaccharide. Moreover, using the energy transfer method, in the pH region higher than the PI of BSA, carboxylic‐type polysaccharides may interact relatively weakly with the binding site II of BSA, but sulfonic‐type ones can selectively interact with binding site I weakly and with binding site II strongly. And in the pH region lower than the PI of BSA, carboxylic‐type polysaccharides interact with binding site II strongly. On the other hand, sulfonic‐type polysaccharides are bound to both binding site I and binding site II very strongly. Copyright © 1999 John Wiley & Sons, Ltd.     

Heterotropic binding of alclofenac and dansylsarcosine to bovine serum albumin

The binding data for the interaction of alclofenac (AF) and dansylsarcosine (DS) to bovine serum albumin (BSA) have respectively yielded nonlinear Scatchard plots. The plots have been subjected to Rosenthal’s method of analysis and thus the ligands have been found to possess two different kinds of sites in BSA. The binding capacities of these sites have been evaluated. The fluorescence competition studies have revealed that the binding of DS to BSA is noncompetitively inhibited by AF. Therefore, the presence of distinct binding sites for AF and DS in BSA could be inferred. The fluorescence quenching studies have also been able to demonstrate this aforesaid fact. The analysis of the quenching data by the modified Stern-Volmer plot has indicated that both the tryptophan (Trp) residues of BSA are accessible to DS for the quenching in absence of AF, but only one of them is accessible in presence of AF. This has led to suggest that the binding site of DS has been in the vicinity of loop 3–4, involving Trp-134 and Trp-212. The binding of AF at a distinct site from that of DS has exerted heterotropic interactions at the DS binding site and thereby inhibited the binding of DS to BSA.     

Investigation on Sonocatalytic Damage to Bovine Serum Albumin (BSA) Under Ultrasonic Irradiation in the Presence of Fe(III)–Citrate Complexes

The interaction of bovine serum albumin (BSA) with Fe(III)?Ccitrate complexes ([Fe^III(cit)(H₂O)₃]^? and [Fe^III(cit)₂]^5?) and the sonocatalytic damage of BSA under ultrasonic irradiation were studied. Additionally, the various factors influencing the sonocatalytic damage of BSA were also studied by means of UV?CVis and fluorescence spectra. The experimental results indicate that the probable fluorescence quenching mechanisms of BSA by Fe(III)?Ccitrate complexes ([Fe^III(cit)(H₂O)₃]^? and [Fe^III(cit)₂]^5?) are both static quenching. Under certain conditions, the degree of damage to BSA is aggravated with increases of ultrasonic irradiation time, Fe(III)?Ccitrate complex concentration, pH value and ionic strength. Moreover, all of the results demonstrate that [Fe^III(cit)₂]^5? displays higher sonocatalytic activity than [Fe^III(cit)(H₂O)₃]^? under the same experimental conditions during the damage process of BSA. Finally, the generation of ·O₂ ^? and ·OH during sonocatalytic processes was estimated using scavengers. Perhaps, the results will be significant for promoting sonodynamic treatment to treat tumors at the molecular level.