Spectroscopic investigation of the interaction between G-quadruplex of KRAS promoter sequence and three isoquinoline alkaloids.

KRAS promoter can form G-quadruplex structure and regulate gene transcription. The drugs which can bind with G-quadruplex of KRAS promoter may be potential remedy for treatment of cancers associated with KRAS mutation. The interaction mechanism between the G-quadruplex of KRAS promoter and three isoquinoline alkaloids (jatrorrhizine, berberine and sanguinarine) has been investigated by UV-visible, fluorescence and circular dichroism spectroscopic methods. The results showed that the three alkaloids can form complexes with G-quadruplex KRAS promoter with the molecular ratio of 1:1, and the binding constants were (0.90±0.16)×106Lmol-1, (0.93±0.21)×106Lmol-1 and (1.16±0.45)×106Lmol-1 for jatrorrhizine, berberine and sanguinarine. The absorption spectra, KI quenching and fluorescence anisotropy and polarization studies suggested jatrorrhizine and berberine interacted with G-quadruplex by not only end-stacking binding mode but also grooves or loops binding mode, while sanguinarine by end-stacking binding mode. Sanguinarine was more beneficial to maintain the stability and parallel conformation of KRAS promoter G-quadruplex. MTT assay was performed to evaluate antiproliferation effects of the three isoquinoline alkaloids on SW620 cells, and the antiproliferation effects of the three alkaloids were sanguinarine > berberine > jatrorrhizine. All the three alkaloids can bind with KRAS promoter G-quadruplex, and sanguinarine had the better binding property and antiproliferation effects on SW620 cells. The results obtained are meaningful to explore potential reagents targeting the parallel G-quadruplex structure of KRAS promoter for gene theraphy of colorectal carcinomas.

[Development of Fluorescence Resonance Energy Transfer Sensor for Determination of Adenosine Monophosphate in Biological Drug].

The biological drug of the calf-blood dialysate has various pharmacological effects. It can promote the oxygen and glucose uptake for the hypoxia cells, and has beneficial effects on the malfunction of the blood circulation and trophic disturbances in the brain, and the impairment of peripheral blood circulation. Furthermore, it is favorable to wound healing and can regulate the central nervous system. Adenosine monophosphate (AMP) is a main active ingredient of the biological drug. In this report, a fluorescence resonance energy transfer (FRET) sensor has been developed with ß-CD-capped ZnS QDs as energy donor and 3-hydroxyflavone (3-HF) as energy acceptor. The results showed that AMP can lead to the fluorescence quenching of the FRET sensor at 526 nm, and the Stern-Volmer curve between the fluorescence quenching and the concentrations of AMP present a satisfactory linearity with the correlation coefficient of 0.996. The developed sensor has successfully applied for determination of the AMP in the biological drug.

Identification and differentiation of the red ink entries of seals on document by laser desorption ionization mass spectrometry.

The establishment of approaches for the differentiation of the ink entries of seals on paper can provide evidence to authenticate the related documents and can play a key role in judicial expertise. The identification and discrimination method for 38 red ink entries of seals on paper has been investigated using laser desorption ionization mass spectrometry (LDI-MS). Six dye components for the ink pastes of seals, Scarlet powder (SP), Bronze Red C (BR), Fast Red R (FR), Basic Violet 3 (BV3), Pigment Red 22 (PR22) and Pigment Red 112 (PR112), have been identified by their LDI-MS spectra, and the results have been confirmed by electrospray ionization quadruple-time of flight mass spectrometry (QTOF-ESI-MS/MS) and inductively coupled plasma atomic emission spectrometry (ICP-AES). The 38 ink entries were classified into six groups based on the presence or the absence of the pigments in their positive and negative LDI-MS spectra, and the discrimination power (DP) was calculated to be about 82%. The ink entries within each group were further differentiated from the relative peak areas (RPA) of the fragments for the pigments and the profile of their LDI-MS spectra, and thus the DP was increased to 98%. All the 38 ink entries could be discriminated (the DP was 100%), if including the contribution of unknown peaks. Compared with the results obtained by the FTIR and Raman methods, the established LDI-MS approach could provide more information of the dye components in the ink entries. The results showed that the developed LDI-MS method is powerful, sensitive and rapid and can directly differentiate the red ink entries of seals from paper substrates, thus offering a novel approach to judge the authenticity of documents.

Surface molecularly imprinted polymers with synthetic dummy template for simultaneously selective recognition of nine phthalate esters.

Dummy template molecularly imprinted polymers (DMIPs) on silica gel particles for simultaneously selective recognition of nine phthalate esters have been prepared. A novel dummy template molecule with similar structural skeleton to the phthalate ester, diethyl N,N'-phthaloyl-bis(11-aminoundecanoate), has been designed and synthesized. The DMIP films were grafted on the surface of silica gel particles by a sol-gel process with 3-aminopropyltriethoxysilane (APTES) and tetramethoxysilane (TEOS) as functional monomer and cross-linker, respectively, and the obtained sorbents have been characterized by FTIR with diffuse reflectance accessory. The maximum static adsorption capacities of the DMIPs and NIP sorbents for the nine phthalate esters were 281 and 132mg/g respectively, and the results of dynamic adsorption showed that the adsorption equilibrium could be achieved about 5min for the DMIPs sorbents. The imprint factors of the sorbents ranged from 1.8 to 3.0 for eight of the phthalate esters except for Diamyl phthalate, which indicated that the DMIPs sorbents have high selectivity. The competitive experiments of the nine phthalates with some of their analogues on the sorbents illustrated that the DMIPs sorbents have high specificity for the phthalates. A GC-MS method for determination of the phthalate residues in fruit juice have been developed with the DMIPs as sorbents for the solid phase extraction (SPE) in the sample pretreatment procedures. The spiking recoveries of the phthalates were in the range of 72-100.2% with relative standard deviations lower than 10.2%. The results indicated that the obtained sorbents could specifically recognize the phthalates from complex matrices, which provide a new train of thought for preparing the DMIPs sorbents.

Simultaneous determination of ten steroid hormones in animal origin food by matrix solid-phase dispersion and liquid chromatography-electrospray tandem mass spectrometry.

An UPLC-MS/MS method for determination of ten steroid hormones in animal origin food has been developed with pretreatment of the samples by matrix solid-phase dispersion (MSPD). The MSPD conditions, including the dispersing sorbents, elution solvents, ratio of sorbent to sample and the volume of the elution solvent have been investigated and optimised, and the method has been evaluated and validated. The results showed that the developed method has satisfactory linearity between the MS/MS responses of the analytes and the concentration of the steroid hormones, and the limits of the detection can reach 0.01µg/kg for most of the analytes. The spiking recoveries of the steroid hormones in chicken, pork, beef and sausage samples were between 76.8% and 98.7% with RSDs lower than 10%. The results demonstrated that the developed approach has high sensitivity and repeatability, and can rapidly determinate the trace residues of steroid hormones in complex food matrices.

An immunosensor based on magnetic relaxation switch and polystyrene microparticle-induced immune multivalency enrichment system for the detection of Pantoea stewartii subsp. Stewartii.

A rapid, sensitive, and simple immunosensor has been developed for the detection of Pantoea stewartii subsp. Stewartii (Pss). This immunosensor combines magnetic relaxation switch (MRS) assay with polystyrene microparticle-induced immune multivalency enrichment system. Comparing to conventional enzyme-linked immunosorbent assay (ELISA), the immunosensor developed in this study provides higher sensitivity and requires less analysis time. The detection limit of Pss obtained by immunosensor was determined to be 10(3)cfu/mL, 50 times lower than that by ELISA (5×10(4)cfu/mL), while the analysis time required by immunosensor is 30min much shorter than that by ELISA. The average recoveries studied with Pss at various spiking levels ranged from 85.5% to 93.4% with a relative standard deviation (RSD) below 6.0%. No cross-reaction with the other five strains was found, demonstrating a good specificity of Pss detection. The results showed that the MRS immunosensor combined with PS-induced immune multivalency enhancement system is a promising platform for the determination of large biological molecules due to its high sensitivity, specificity, homogeneity, and speed.

Evidence of different G-quadruplex DNA binding with biogenic polyamines probed by electrospray ionization-quadrupole time of flight mass spectrometry, circular dichroism and atomic force microscopy.

The binding properties of five G-quadruplex oligonucleotides (humtel24, k-ras32, c-myc22, c-kit1 and c-kit2) with polyamines have been investigated by electrospray ionization-quadrupole time of flight mass spectrometry, circular dichroism, melting temperature, atomic force microscopy (AFM) and molecular simulation. The MS results demonstrated that the polyamines and G-quadruplex DNA can form complexes with high affinity, and one molecule of G-quadruplex DNA can combine several molecules (1-5) of polyamines. The binding affinities of the polyamines to DNA were in the order of spermine > spermidine > putrescine. After binding with polyamines, the conformations of the G-quadruplex DNA were significantly changed, and spermine can induce the configurations of k-ras32 and c-kit1 to deviate from their G-quadruplex structures at high concentrations. In the presence of K(+), the conformations of G-quadruplex DNA were stabilized, while polyamines can also induced alterations of their configurations. Melting temperature experiments suggested that the Tm of the DNA-polyamine complexes obviously increased both in the absence and presence of K(+). The AFM results indicated that polyamines can induce aggregation of G-quadruplex DNA. Above results illustrated that the polyamines bound with the phosphate backbone and the base-pairs of G-quadruplex structures. Combining with the molecular simulation, the binding mode of the G-quadruplex DNA and polyamines were discussed. The results obtained would be beneficial for understanding the biological and physiological functions of polyamines and provide useful information for development of antitumor drugs.

Immunosensor based on magnetic relaxation switch and biotin-streptavidin system for the detection of Kanamycin in milk.

A rapid, sensitive, and simple immunosensor was developed for the detection of Kanamycin (KM) in milk. This immunosensor is based on magnetic relaxation switch (MRS) assay and biotin-streptavidin system (B-SA system). The target analyte (KM) competed with those on the surface of the superparamagnetic iron oxide (SPIO) nanoparticles and hence affected the formation of SPIO aggregates. The dispersed and aggregated states of SPIO can modulate the spin-spin relaxation time (T(2)) of the neighboring water molecule. T(2) was then changed as an effect of the target analyte. The B-SA system was used to amplify the SPIO binding, thus enhance the sensitivity. The detection working was 1.5 to 25.2ng mL(-1) and limit of detection (LOD) was determined to be 0.1ng mL(-1). The LOD of the immunosensor decreased tenfold, and its analysis time (45min) was much shorter than that of enzyme-linked immunosorbent assay (6h to 8h). The average recoveries of the KM at various spiking levels ranged from 80.2% to 85.6% with a relative standard deviation (RSD) below 4.0%. The results showed that the MRS immunosensor was a promising platform for the determination of small molecular residues because of its high sensitivity, specificity, homogeneity, and speed.

Quantum dots capped with dummy molecularly imprinted film as luminescent sensor for the determination of tetrabromobisphenol A in water and soils.

Molecularly imprinted film with diphenolic acid (DPA) as dummy template molecule has been grafted on the surface of Mn-doped ZnS quantum dots (QDs) to develop a selective and sensitive sensor for rapid determination of tetrabromobisphenol A (TBBPA) in water and soils. The obtained DPA-MIP-QDs sensor has distinguished selectivity and high binding affinity to TBBPA. The fluorescence quenching fractions of the sensor presented a satisfactory linearity with the concentrations of TBBPA in the range of 0.1-100 µM, and its limit of detection can reach 0.015 µM. The sensor has been successfully applied to determine the TBBPA in water and soil samples, and the average recoveries of the TBBPA at various spiking levels ranged from 80.2% to 96.5% with relative standard deviation below 8.0%. The results provided a clue to develop sensors for rapid determination of hazardous materials from complex matrixes.

Nondestructive identification for red ink entries of seals by Raman and Fourier transform infrared spectrometry.

Determination of the red ink entries of seals on documents can provide valuable evidences for solving related crimes, distinguishing the truth of artworks, and so establishment of nondestructive approaches would play a key role in forensic analysis and related aspects. Raman and FT-IR spectroscopy have been applied for analyzing 105 kinds of red ink entries on documents. The dye components of the ink entries were identified by FT-Raman and confocal Raman microspectroscopy, and then the ink entries were classified into four groups based on these dye components. The ink entries were further discriminated by their FT-IR spectra according to adsorption peaks of the main components, the relative intensities of the characteristic bands and the profiles of the spectra. The results showed that 70 ink entries out of 105 have been individually identified and the remaining 35 ink entries can be divided into 13 subclasses. Combination of Raman and FT-IR spectroscopic methods can provide a powerful nondestructive discriminating tool for identification of the red ink entries of seals on papers. These approaches would have potential application in archeology, art and forensic science.

[Interaction between three isoflavones and different isomers of human serum albumin].

The interaction mechanisms between isoflavones (Genistein, 3', 4', 7-trihydroxyisoflavone and Biochanin A) and different isomers of human serum albumin (HSA) were investigated by fluorescence spectroscopy. Various parameters (quenching rate constants, binding constants and number of binding sites) of isoflavones-human serum albumin complexes were calculated. The results showed that the isoflavones have only one binding site on human serum albumin, located at the Site I, and the binding constants were between 0.17 x 10(5) and 1.20 x 10(5) L x mol(-1). Fluorescence enhancement experiments showed that the fluorescence intensities of the drugs significantly increased after interacting with HSA, indicating that the combination of drug and HSA had occurred. The binding mechanisms between three isoflavones and HSA were discussed.

Spectroscopic investigation on the interaction of 3,7-dihydroxyflavone with different isomers of human serum albumin.

The interaction mechanism of 3,7-dihydroxyflavone (3,7-diHF) and human serum albumin (HSA) was investigated by fluorescence quenching, fluorescence enhancement, steady-state and time-resolved fluorescence emission and UV-vis absorption spectrometry. The binding site of 3,7-diHF on protein was determined by investigating the spectroscopic properties of 3,7-diHF-HSA complex at pH 7.4 and pH 3.5 individually, and confirmed by the site marker competitive experiments. The binding parameters of 3,7-diHF-HSA complex were estimated by fluorescence quenching experiments, and the data were in good agreement with the results obtained from fluorescence enhancement measurements. A remarkable increase in the fluorescence anisotropy values suggested that 3,7-diHF bound at a motional restricted pocket on HSA. The results indicated that 3,7-diHF, in anionic form, was bound within the hydrophobic pockets of the subdomain IIA of HSA (site I), and stabilised mainly by electrostatic force and ionic interactions. The binding mode of drug-protein was discussed based on above experimental results.

Dummy molecularly imprinted polymers on silica particles for selective solid-phase extraction of tetrabromobisphenol A from water samples.

Surface molecular imprinted polymers (MIPs) on silica gel particles for highly selective recognition of tetrabromobisphenol A (TBBPA) were prepared by a sol-gel process. Diphenolic Acid (DPA) and bisphenol A (BPA), whose structures were similar to that of TBBPA were selected as dummy template molecules, and 3-aminopropyltriethoxysilane (APTES) and tetramethoxysilane (TEOS) were chosen as functional monomer and cross-linker, respectively. The obtained materials were characterized by FT-IR with diffuse reflectance accessory and the results indicated polymers were successfully grafted on the surface of silica gel supporters. The maximum static adsorption capacities for TBBPA of the DPA-MIPs, BPA-MIPs and non-imprinted polymers (NIPs) were 45, 38 and 22 mg g(-1) respectively, and the results of dynamic adsorption showed that the adsorption equilibrium can be achieved within 15 min for DPA- and BPA-MIPs. Both the DPA- and BPA-MIPs have higher selectivity for TBBPA than that of NIP when they are used as the sorbents for the solid phase extraction (SPE), while the adsorption property of DPA-MIPs was superior to that of BPA-MIPs at low concentration levels of TBBPA. The results indicated DPA-MIPs had more high affinity binding sites for TBBPA, which demonstrated that the strong interactions between the template and the functional monomer were favorable to form high affinity binding sites and improve the selectivity of the polymers. A corresponding analytical method for determination of the TBBPA residues in environmental samples was developed. The recoveries of TBBPA in tap water, river water and lake water were in the range from 85% to 97% with relative standard deviations below 7%, and its limit of detection can reach 2 ng mL(-1).

Determination of emulsion explosives with Span-80 as emulsifier by gas chromatography-mass spectrometry.

A novel approach for identification and determination of emulsion explosives with Span-80 (sorbitol mono-oleate) as the emulsifier and their postblast residues by gas chromatography-mass spectrometry (GC-MS) has been developed. 24 kinds of emulsion explosives collected have been processed by transesterification reaction with metholic KOH solution and the emulsifier has turned into methyl esters of fatty acids. From the peak area ratios of their methyl esters, most of these emulsion explosives can be differentiated. In order to detect the postblast residues of emulsion explosives, the sorbitols in the emulsifier Span-80 obtained after transesterification reaction have been further derivatized by silylation reaction with N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS) as the derivatizing reagent. The derivatization conditions were optimized and the derivatives were determined by GC-MS. The results showed that the silylation derivatives of sorbitol and it isomers, combined with hydrocarbon compounds and methyl esters of fatty acids, were the characteristic components for identification of the emulsion explosives. The established approach was applied to analyze the postblast residues of emulsion explosives. It has been found that the method was sensitive and specific, especially when detecting the derivatives of sorbitol and its isomers by GC-MS in selecting ion mode. The information of the characteristic components can help probe the origin of the emulsion explosives and providing scientific evidences and clues for solving the crimes of the emulsion explosive explosion.

[Spectroscopic investigation on the interaction of protocatechuic acid and veratric acid with biomacromolecules].

Protocatechuic acid (P) and veratric acid (V) are phenolic acidic compounds and have a wide biological and pharmaceutical activities, and their interaction with biomacromolecule has been a hot topic. The interaction mechanism of P and V with fsDNA was investigated by fluorescence and UV absorption spectroscopic methods. The UV results showed that P and V have three strong absorption bands at 190-230 nm (K band), 230-270 nm (B band) and 270-310 nm (R band) respectively. When the excitation wavelength was 280 nm, the fluorescence emission bands of P and V were at 338 and 334 nm, respectively, while the fluorescence emission band of DNA was very weak and had little influence on those of the P and V. The fluorescence intensities of the P and V were strongly quenched after interacting with fsDNA, and their Stern-Volmer quenching rate constants were 1.03 x 10(12) and 0.61 x 10(12) L x mol(-1) x s(-1), respectively. It was illustrated that the fluorescence quenching was mainly static and the complex was formed between the drug and fsDNA. When the concentration of DNA was high, their Stern-Volmer curves were not linear, and it was indicated that the quenching mechanism was complex and may contain dynamic quenching process. Their binding constants were calculated based on the static fluorescence quenching, with K(fsDNA/P) = 6.22 x 10(6) L x mol(-1) and K(fsDNA/v) = 1.57 x 10(4) L x mol(-1). The investigation showed that the molecular ratio of V-fsDNA was 1 : 1, while that of P-fsDNA was 1: 2. It was demonstrated that the protocatechuic acid can bind with two bases of fsDNA, which was related to the two hydroxyl groups on the drug molecule. The results showed that the structure of P and V greatly influenced their binding mode with DNA molecules.

Differentiation and dating of red ink entries of seals on documents by HPLC and GC/MS.

A novel approach for differentiation and dating of red ink entries of seals on documents was developed based on ion-pairing HPLC (IP-HPLC) and GC/MS. Sixty-nine red ink pastes of seals were collected and the chromatographic conditions for separation of the dye components by IP-HPLC and the volatile additives by GC/MS in the ink entries were optimized. According to the dye components and additives, the ink entries were classified by HPLC with a multi-wavelength UV detector. The volatile components of the inks were identified by GC/MS and the classification of the ink entries was also investigated based on these volatile additives. The results showed that most of the ink entries of the seals can be differentiated by combining HPLC with a multi-wavelength detector and GC/MS methods. The degradation of the standard dye mixtures and the compositional changes of the ink entries of seals were investigated in light or natural aging conditions. The results indicated that the dye components decomposed in light or natural storage conditions, while the rates of the degradation depended on the structures of the dye components, the aging conditions, even the additives of the ink pastes. The results also showed that there existed good relationships between the compositional changes of the ink entries and the aging time, which can provide scientific evidences and valuable clues for dating of the ink entries.

Identification and dating of the fountain pen ink entries on documents by ion-pairing high-performance liquid chromatography.

A novel approach for the identification and dating of the fountain pen ink entries on paper has been established by ion-pairing high-performance liquid chromatography (IP-HPLC). Twelve black and six red fountain inks have been collected, and their ink entries have been prepared by drawing lines on paper. The chromatographic conditions for separation of their dye components after extraction with solvents were optimized. Under the optimized conditions, the 18 fountain pen inks were differentiated individually by comparing the number of detectable main or minor dye components, and the relative peak intensities of each component. The ink entries were artificially and naturally aged, and the analysis results showed that the ink dye components were significantly decomposed when exposed to UV or fluorescent light compare to those of inks stored under natural condition. The changes of the relative peak height for the dye components were linearly related to the aging time, especially under natural aging conditions. The degradation characteristics of the dye components under different aging conditions provide scientific evidences for dating of the suspicious fountain pen ink entries on document.

Application of matrix solid-phase dispersion and high-performance liquid chromatography for determination of sulfonamides in honey.

A novel method for simultaneous determination of 8 sulfonamide residues (sulfathiazole, sulfapyridine, sulfadiazine, sulfamerazine, sulfamonome-thoxine, sulfachloropyridazine, sulfamethoxazole, and sulfadimethoxine) in honey samples by high-performance liquid chromatography (HPLC) has been developed on the basis of precolumn derivatization with 9-fluorenylmethyl-chloroformate (FMOC-Cl). Sulfonamide residues in honey samples were extracted and purified by matrix solid-phase dispersion with C18 as the solid support. The residues were derivatized by FMOC-CI, and the FMOC-sulfonamide derivatives were further purified by solid-phase extraction with silica gel as the solid support prior to HPLC analysis. The average recoveries for most sulfonamide compounds at different spiking levels (from 10 to 250 microg/kg) were > 70% with relative standard deviations < 16%, and their limits of detection were 4.0 microg/kg. The established analytical method has high sensitivity and repeatability and can be applicable for determining the sulfonamide residues in various honey matrixes.

Determination of sulphonamides in animal tissues by high performance liquid chromatography with pre-column derivatization of 9-fluorenylmethyl chloroformate.

A novel approach for simultaneous determination of 12 sulphonamides (sulphadiazine, sulphamethazine, sulphathiazole, sulphadimethoxine, sulphamerazine, sulphapyridine, sulphamethoxazole, suphamethizole, sulphaquinoxaline, sulphameter, sulphamonomethoxine, and sulphachloropyridazine) in animal tissues (swine muscle and liver, chicken muscle, beef muscle) by HPLC with UV detection has been developed. A pre-column derivatization of the sulphonamide compounds with 9-fluorenylmethyl chloroformate (FMOC-Cl) has been proposed and the reaction conditions have been optimized. The FMOC-sulphonamide derivatives were purified by SPE with silica gel as solid support prior to HPLC separation. The limits of detection for the sulphonamide compounds were greatly improved after the derivatization and purification step for the derivatives. Sulphonamide residues in animal tissues were extracted by acetonitrile and purified by solid phase extraction with C(18) as the solid support. The method developed has high sensitivity and good repeatability, and the average recoveries for most of the sulphonamides at various spiking levels were above 70% with relative standard deviations below 13.7%. The limits of detection for most sulphonamides can reach 3-5 microg/kg.

Characterization of the myricetin-human serum albumin complex by spectroscopic and molecular modeling approaches.

The interaction mechanism of flavonol myricetin (3,5,7,3',4',5'-hexahydroxyflavone) and human serum albumin (HSA) has been characterized by fluorescence, electronic absorption, and Fourier transform infrared (FT-IR) spectroscopic approaches and the molecular modeling method. The structural characteristics of myricetin and HSA were probed, and their binding affinities were determined under different pH conditions. The results showed that the binding abilities of the drug to protein decreased under lower pH conditions (pH 3.5 and 2.0) due to the alterations of the protein secondary and tertiary structures. The second derivative absorption spectra of myricetin after interacting with the protein showed that the drug existed as an anion form in the binding pocket. The fluorescence emission intensities of the normal and excited-state proton transfer (ESTP) tautomer of myricetin significantly enhanced in the presence of HSA with conspicuous shifts of the emission bands when excited with a wavelength of 370 nm, while the intensity ratios of the normal to ESTP tautomers rose rapidly with the increase of the HSA concentrations under different pH environments. This illustrated that the fluorescence emission of the normal tautomer (S1-S0, non-proton-transferred) predominated due to the interaction of drug and surrounding polar and ionic side chains of amino acid residues in the binding cavity. The similar spectroscopic properties of myricetin-HSA complex at pH 7.4 and 3.5 showed that the drug was located in subdomain IIA of the protein in the vicinity of the single Trp 214 because of the unfolding of the protein domain III in its F state. From the molecular modeling results, the drug-protein complex was stabilized by electrostatic force and hydrogen bonding with the amino acid residue in the binding pocket, which was consistent with the experimental results.