Copper-1,10-phenanthroline (phen) complex [Cu(phen)2] has been typically known as DNA-cleaving agent. And now it becomes more important for developing multifunctional drugs with its improved cytotoxic properties. In our study, we probed the cytophysiological mechanism of Cu(phen)2. HepG2 cells were more sensitive to Cu(phen)2 with an IC50 of 4.03 μM than other three kinds of cell lines. After treated by Cu(phen)2, HepG2 cells had some typical morphological changes which happened to its nucleus. DNA ladder’s occurence and Annexin V-positive increased cells indicated that Cu(phen)2 induced HepG2 cells into apoptosis. Further studies showed that Cu(phen)2 treatment resulted in significant G2/M phase arrest and collapse of mitochondrial membrane potential. Several cell cycle-related factors were down-regulated, including Cyclin A, Cyclin B1 and Cdc2. But p21 and p53 were up-regulated. DNA damage, microtubule disorganization and mitotic arrest through spindle assembly checkpoint activation were observed in Cu(phen)2-treated cells. The activation of caspase-3, 8 & 9 were checked out. The increased-expression ratio of Bax/Bcl-2 was detected. The expression levels of Bcl-xL and Bid were found to decrease. These meant that a mitochondrial-related apoptosis pathway was activated in treated HepG2 cells. Furthermore, some ER stress-associated signaling factors were found to be up-regulated, such as Grp78, XBP-1and CHOP. Ca2+ was also found to be released from the ER lumen. Collectively, our findings demonstrate that Cu(phen)2 induces apoptosis in HepG2 cells via mitotic arrest and mitochondrial- and ER-stress-related signaling pathways.

To study the thermostability of Nattokinase (subtilisin NAT, NK), three double mutant plasmids (pET-28a-NKG61C/S98C, pET-28a-NKT22C/S87C, pET-28a-NKS24C/S87C) were constructed by site-directed mutagenesis. Target enzymes were detected using SDS-PAGE and disulfide bond formation was detected using Western blotting analysis. Thermostability was tested by rates of inactivation at certain temperature. The results showed that disulfide bond was not formed within two cysteines and the thermostability of three double mutants was not increased compared with the wild-type NK. The thermostability of NK performed in Ca2+ was stronger than in ethylenediaminetetraacetic acid (EDTA). But when the temperature reached 62 °C, the enzymes rapidly denatured and inactivated even in the presence of Ca2+. Although the thermostability of mutants was not increased, this study shows a tendency of improving thermostability of NK in protein engineering.

The binding reaction between docetaxel (DTX) and human hemoglobin (HHb) was investigated systematically with various spectroscopic methods including fluorescence quenching technique, ultraviolet (UV)–vis absorption, synchronous fluorescence, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. Analysis of fluorescence data showed that the quenching mechanism was the dynamic quenching and each protein had only one binding site for the drug. Two thermodynamic parameters, the enthalpy change and the entropy change were calculated to be 9.18 kJ mol−1 and 116 J mol−1 K−1, respectively, which suggested that hydrophobic interaction played a major role in the binding reaction. The results from different spectroscopic methods also showed that DTX could induce conformational changes of HHb. The molecular docking simulation demonstrated that DTX was located in the central cavity of HHb.Highlights► We study the interaction between DTX and HHb by different spectroscopic techniques. ► The quenching mechanism was a dynamic quenching. ► There was only one binding site on HHb for DTX. ► Thermodynamic parameters suggested that hydrophobic interaction was a major force. ► The conformation of protein changed was induced by DTX.

Pharmaceutical interactions with human serum albumin (HSA) are of great interest, because HSA is a pharmacokinetic determinant and a good model for exploring the protein−ligand interactions. Due to their hydrophobic nature, naturally occurring flavones, which possess various pharmacological activities, bind to HSA in human plasma. Here, we have identified the binding modes of two representative flavones—baicalin (BLI) and its aglycon, baicalein (BLE)—to HSA using a combination of experimental and computational approaches. The association properties were measured by applying spectroscopic methods, and a higher affinity was found for BLE. As evidenced by displacement and chemical unfolding assays, both ligands bind at Sudlow site I. Furthermore, molecular docking was utilized to characterize the models of HSA−flavone complexes, and molecular dynamics (MD) simulations as well as free energy calculations were undertaken to examine the energy contributions and the roles of various amino acid residues of HSA in flavones binding; the mechanism whereby glycosylation affects the association was also discussed. The present work provides reasonable binding models for both flavones to HSA.

The slightly water-soluble cancer chemopreventive polyphenol resveratrol (Res) and its inclusions with β-cyclodextrin (β-CD) and hydroxypropyl-β-cyclodextrin (HP-CD) have been investigated. The stoichiometric ratios and stability constants have been determined by phase-solubility measurements. In all cases 1:1 complexes are formed. The inclusion ability of HP-CD is larger than that of β-CD. The antioxidant activity of the Res complexes has been determined by the scavenging of the stable radical DPPH. The scavenging capacity of the two complexes increases with increasing concentration of cyclodextrins. Res/HP-CD complex shows a higher scavenging capacity than Res/β-CD complex. The antioxidant activity of Res in free form has little difference with Res in complexed form at the same concentration.

Docetaxel is a semi-synthetic product derived from the needles of the European yew. It is an antineoplastic agent belonging to the taxoid family. The interaction between docetaxel and human serum albumin (HSA) has been investigated systematically by the fluorescence quenching technique, synchronous fluorescence spectroscopy, ultraviolet (UV)–vis absorption spectroscopy, circular dichroism (CD) spectroscopy and Fourier transform infrared (FT-IR) under physiological conditions. Our fluorescence data showed that HSA had only one docetaxel binding site and the binding process was a static quenching procedure. According to the Van’t Hoff equation, the thermodynamic parameters standard enthalpy (ΔH0) and standard entropy (ΔS0) were calculated to be −41.07 KJ mol−1 and −49.72 J mol−1 K−1. These results suggested that hydrogen bond was the predominant intermolecular force stabling the docetaxel–HSA complex. The data from the CD, FT-IR and UV–vis spectroscopy supported the change in the secondary structure of protein caused by the interaction of docetaxel with HSA.

Mitomycin C (MMC) was the first recognized bioreductive alkylating agent, and has been widely used clinically for antitumor therapy. The binding of MMC to two human blood proteins, human serum albumin (HSA) and human hemoglobin (HHb), have been investigated by fluorescence quenching, synchronous fluorescence, circular dichroism (CD) spectroscopy and molecular docking methods. The fluorescence data showed that binding of MMC to proteins caused strong fluorescence quenching of proteins through a static quenching way, and each protein had only one binding site for the drug. The binding constants of MMC to HSA and HHb at 298 K were 2.71 × 104 and 2.56 × 104 L mol−1, respectively. Thermodynamic analysis suggested that both hydrophobic interaction and hydrogen bonding played major roles in the binding of MMC to HSA or HHb. The CD spectroscopy indicated that the secondary structures of the two proteins were not changed in the presence of MMC. The study of molecular docking showed that MMC was located in the entrance of site I of HSA, and in the central cavity of HHb.

In present work the complexation of Res with two kinds of cyclodextrins (CDs), native β-cyclodextrin (β-CD) and modified hydroxypropyl-β-cyclodextrin (HP-CD), have been investigated by fluorescence spectroscopy, 1H-NMR spectroscopy and molecular modeling methods. The stoichiometric ratios, inclusion constants and thermodynamic parameters have been determined by the fluorescence data. In all cases 1:1 inclusion complexes are formed. The inclusion ability of HP-CD is larger than that of β-CD. Both inclusion processes have negative ∆G, negative ∆H and positive ∆S. Thermodynamic analysis suggests that Van der Waals force of guest-host interactions and the release of high-enthalpy water molecules from the cavity of CDs play important roles in driving complex formation. The study of molecular modeling shows that part of the A-ring and the B-ring of Res are placed in the cavity of β-CD, and the hydroxyl groups are projected outside. As for Res in HP-CD, the B-ring of Res is included in the cavity of HP-CD, and part of the A-ring is pointed outside. 1H-NMR spectroscopy results show that H2, H3, H4 and H5 protons of Res are more affected by the complexatin, indicating that they are located inside the torus of CDs, which are in agreement with the result of the molecular modeling.

A new biocatalyst route for the synthesis of a conducting polyaniline (PANI)/lignosulfonate (LGS) complex was presented. Four different catalysts such as hemoglobin (Hb), 5, 10, 15, 20-tetrakis (meso-hydroxyphenyl) porphyrin, iron (II) tetrasulfophthalocyanine and ferric chloride were used to polymerize aniline in the presence of a natural polyelectrolytes template LGS. The experimental results show that Hb is an effective catalyst in this case and the synthesis is simple, and the conditions are mild in that the polymerization may be carried out in lower pH (1.0–4.0) buffered solution and optimal pH of 2.0. Varying concentrations of aniline, LGS and H2O2 in feed the favorable conditions for the production of PANI were determined. UV-vis absorption, FTIR, elemental analysis, conductivity, cyclic voltammetry and thermogravimetric analyses confirm the formation of thermally stable and electroactive PANI.

The thermal unfolding of Urinary Trypsin Inhibitor (UTI) was studied by several methods: Circular Dichroism (CD), Fluorescence and UV–Vis spectra. Thermal melting of UTI, dissolved in the neutral and basic buffers, was proved to be irreversible and two domains of UTI unfolded simultaneously, but the melting was reversible and the intermediate was observed when pH is lower than 4.2. The result suggested that heat and changes in pH, which had a more important impact on the stabilization of the domain I and the interaction between two domains, might cause different unfolding transitions. A reasonable explanation was deduced for the mechanism of reversible and irreversible thermal unfolding based on the effect of pH on the protein structure, the analysis of thermal transitions and the result of Electron Microscopy: In neutral and basic buffers, the Reactive Central Loop (RCL) in domain II can interact with or insert into the partial expanding domain I and UTI become self-polymerization, however, no aggregation can be observed in acid buffer since low pH and heat destabilized the structure of the domain I and the native conformation can restructure. The interaction between the special structural element RCL and domain I play an important role in the formation of polymer which was different from other two reasons given by other authors—the cleavage of disulfide and the formation of irregular polymer mainly based on hydrophobic interaction.

Essential oil of the subterranean part of Cacalia tangutica (Maxim.) Hand.-Mazz was analyzed by gas chromatography (GC)-mass spectrum (MS) technique in two different capillary columns of different polarities. Thirty-one components were identified in the oil and the main compounds were α-zingiberene (13.49%), germacrene D (10.76%), α-pinene (8.54%), caryophyllene(Z-) (6.36%), linalool (6.16%), β-myrcene (4.89%), β-ocimene (Z-) (4.40%)and ocimenone(Z-) (3.58%). The antimicrobial activity of the oil was evaluated against 2 fungi and 12 bacteria including 6 clinically isolated strains using the agar disc diffusion and broth microdilution methods. The results show that the oil presented a broad antimicrobial spectrum and had better antimicrobial activity against yeast and gram-positive bacteria. The minimum inhibitory concentration values were 0.16–5.00 g/L and minimum bactericidal concentration values were 0.16–5.00 g/L.

DNA is the potential target of numerous drugs utilized widely in clinical cancer therapy. Here we employed bleomycin A5, with its deactivated form as contrast, to investigate the interaction between small pharmaceutical and DNA. Electrophoresis mobility shift assay (EMSA) which is a common technique used in studying specific interactions between DNA and proteins is applied in visualizing the binding of bleomycin A5 with DNA intuitively. More accurate association equilibrium constant for native and deactivated bleomycin A5 to DNA achieved on biosensor IAsys AUTO+ is 1.25 × 104 and 1.3 × 103 M−1, respectively. With combination of EMSA and biosensor, a qualitative and quantitative method is described, which can be extended to studying the binding of most small molecules with targeting DNA and serves as a powerful tool in designing and screening for novel drugs.

The leaf essential oil of Photinia serrulata was obtained by hydrodistillation and analyzed by GC and GC-MS. Seventy-one components were identified in the essential oil and the main components of the oil were 10-epi-γ-eudesmol (12.72%), pinene (6.85%), sabinene (5.93%), α-humulene (5.87%) and α-thujene (5.47%). The in vitro cytotoxicity of the oil on human cancer cell lines Hela, A-549 and Bel-7402 was examined. The oil was found to be very active against all the three human tumor cell lines tested with low IC50 of 0.0427 μl/ml (Hela), 0.0219 μl/ml (A-549) and 0.0301 μl/ml (Bel-7402). The oil was also found to possess antioxidant activity as demonstrated by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical method.

The mechanism of cytotoxicity on liver carcinoma Bel-7402 cells induced by copper-1,10-phenanthroline, Cu(OP)2, has been studied. Cell viability and apoptotic rate were examined in cells treated with Cu(OP)2 or Cu2+ alone. It was found that the apoptosis induced by Cu(OP)2 could not be induced by Cu2+ or OP alone in our experimental conditions. Total copper content in cells was measured by atomic absorption spectrophotometry, and the abnormal elevation of intracellular copper transported by lipophilic OP ligand may play the role of initial factor in the apoptosis, which caused subsequent redox state changes in cells. Intracellular levels of reactive oxygen species (ROS) were detected by fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Reduced (GSH) and total glutathione (GSSG + GSH) were determined by High-performance liquid chromatography (HPLC) after derivatization, and the ratios of GSH/GSSG were subsequently calculated. The overproduction of ROS and the decreased GSH/GSSG ratio were observed in cells which represented the occurrence of oxidative stress in the apoptosis. Oxidative DNA damage was also found in cells treated with Cu(OP)2 in the early stage of the apoptosis, and it suggests that the activation of DNA repair system may be involved in the pathway of the apoptosis induced by Cu(OP)2.

Resveratrol is a natural phytoalexin with pharmacologic effects on several human diseases: carcinogenesis, coronary heart disease and neurodegenerative disease. Due to its poor water solubility, resveratrol must be bound to proteins to keep it at a high concentration in serum. In our work, the bindings of resveratrol to plasma proteins, human serum albumin (HSA) and hemoglobin (Hb), have been investigated systematically by fluorescence quenching technique, synchronous fluorescence, UV–vis absorption spectroscopy, circular dichroism (CD) spectroscopy and molecular modeling method. The fluorescence data show that the binding of resveratrol to HSA or Hb is a static quenching procedure and each protein has only one binding site for the drug. The binding constant of resveratrol to HSA is larger than that of resveratrol to Hb at corresponding temperature, which indicates that the affinity of HSA toward the drug is higher than that of Hb. The CD spectroscopy indicates that the secondary structures of the proteins are changed in the presence of resveratrol with the reduction of α-helices, which decreased about 18.75% for HSA and 9.43% for Hb at the drug to proteins molar ratio of 2. Thermodynamic analysis and molecular modeling suggest that hydrophobic interaction plays a major role in the binding of resveratrol to HSA, and hydrogen bonding is the mainly binding force in the binding of resveratrol to Hb. The study of molecular modeling shows that resveratrol is located in the hydrophobic cavity between subdomain IB and IIA of HSA (the entrance of site I), or located in the central cavity of Hb (partial to the subunit A).

Daunomycin (DM) is a clinically used antitumor anthracycline antibiotic, which is transported primarily by human serum albumin (HSA) in the blood. Binding characteristics are therefore of interest for both the pharmacokinetics and pharmacodynamics of DM. A new optical biosensor technique based on the resonant mirror was used to characterize interaction of DM with HSA at different temperatures and the affinity constants were obtained. The HSA–DM interaction is exothermic with having favorable enthalpy and entropy followed by the integrated van’t Hoff equation analysis. Fluorescence studies showed that DM has an ability to quench the intrinsic fluorescence of HSA through a static quenching procedure according to the Stern–Volmer equation and DM displays a pH-dependent binding affinity to HSA. Molecular modeling calculations showed that the DM binds HSA to a non-classical drug binding site and further analysis of the binding site of DM within the HSA molecule suggested that hydrophobic contacts, hydrogen bond formation and electrostatic interactions account for the binding of DM.

A three-dimensional structural model of nattokinase (NK) from Bacillus natto was constructed by homology modeling. High-resolution X-ray structures of Subtilisin BPN’ (SB), Subtilisin Carlsberg (SC), Subtilisin E (SE) and Subtilisin Savinase (SS), four proteins with sequential, structural and functional homology were used as templates. Initial models of NK were built by MODELLER and analyzed by the PROCHECK programs. The best quality model was chosen for further refinement by constrained molecular dynamics simulations. The overall quality of the refined model was evaluated. The refined model NKC1 was analyzed by different protein analysis programs including PROCHECK for the evaluation of Ramachandran plot quality, PROSA for testing interaction energies and WHATIF for the calculation of packing quality. This structure was found to be satisfactory and also stable at room temperature as demonstrated by a 300 ps long unconstrained molecular dynamics (MD) simulation. Further docking analysis promoted the coming of a new nucleophilic catalytic mechanism for NK, which is induced by attacking of hydroxyl rich in catalytic environment and locating of S221.

In aqueous neutral sodium dodecyl sulfate (SDS) solution, as meso-tetra(4-hydroxyphenyl)porphyrin (THPP) monomers insert SDS aggregation, a strong porphyrin–porphyrin interaction is observed, and then the porphyrin J-aggregates form slowly. The concentration of SDS necessary to give the maximum amount of J-aggregates is 5.0 mM. The J-aggregates have been studied by optical absorption, fluorescence, and resonance light scattering spectroscopies. The J-aggregates have two strong exciton bands corresponding to the B-band and Q-bands of THPP monomers. The total fluorescence of THPP is quenched due to the aggregate formation. A strong and sharply peaked resonance light scattering signal (at 490 nm) which suggests a delocalized excitonic state is observed just slightly to the red of the J-aggregates absorption maximum. The J-aggregates are still very steady even if the solutions were stored in the dark for more than a year.

J-aggregates of 2-hydroquinone-5,10,15,20-tetra(p-hydroxyphenyl)porphyrin (HQTHPP) induced by N-lauroyl sarcosine (SKL) in aqueous neutral solutions have been studied by optical absorption, fluorescence, and resonance light-scattering spectroscopies. As SKL concentration increases, the spectra evolve to reveal the presence of four independent species with relative concentration. The most important species is J-aggregates. The J-aggregates have two strong exciton bands corresponding to the B-band and Q-bands of HQTHPP monomers, and are found to be stable when the surfactant concentration is below 8.0 mmol/L. But above 8.0 mmol/L, the J-aggregates dissolve gradually into another species: porphyrin monomers. The total fluorescence of HQTHPP is quenched due to the aggregate formation. A strong and sharply peaked resonance light-scattering signal (>1800 counts/s, centered at 490 nm) is observed just slightly to the red of the J-aggregate absorption maximum. In the case of cetyltrimethyl-ammonium bromide, increasing surfactant concentrations have only shown to favor solubilization of porphyrin monomers. Evidently, the nature of polar headgroups of surfactants influences the tendency of THPP to aggregate.

The essential oil of Scutellaria barbata was obtained by hydrodistillation with a 0.3% (v/w) yield and analysed by GC and GC-MS. The main compounds in the oil were hexahydrofarnesylacetone (11.0%), 3,7,11,15-tetramethyl-2-hexadecen-1-ol (7.8%), menthol (7.7%) and 1-octen-3-ol (7.1%). The antimicrobial activity of the oil was evaluated against 17 microorganisms using disc diffusion and broth microdilution methods. The gram-positive bacteria, including methicillin-resistant Staphlococcus aureus, were more sensitive to the oil than gram-negative bacteria and yeasts.The essential oil of Scutellaria barbata has been studied. Forty-one components were identified. The antimicrobial activity of the oil was evaluated against 17 microorganisms, including bacteria and yeast. The gram-positive bacteria were the most sensitive.

The effects of copper-1,10-phenanthroline combined with hyperthermia on human liver hepatoma cell line, Bel-7402 were studied. The effect was evaluated by the mean thermal power of the cells and total energy Q produced during the measurement period (35 h). It was found that the energy produced reduced after the treatment. Condensation of nuclear chromatin and apoptotic bodies can be observed from fluorescence microscope, which showed apoptosis occurs under the action of copper-1,10-phenanthroline combined with hyperthermia. The analysis by flow cytometry showed the proportion of apoptotic cells in the cell population increased. It indicates that the combination of hyperthermia and copper-1,10-phenanthroline has a synergetic effect on Bel-7402.

Here, we show that during in vivo folding of the precursor, the propeptide of subtilisin nattokinase functions as an intramolecular chaperone (IMC) that organises the in vivo folding of the subtilisin domain. Two residues belonging to β-strands formed by conserved regions of the IMC are crucial for the folding of the subtilisin domain through direct interactions. An identical protease can fold into different conformations in vivo due to the action of a mutated IMC, resulting in different kinetic parameters. Some interfacial changes involving conserved regions, even those induced by the subtilisin domain, blocked subtilisin folding and altered its conformation. Insight into the interaction between the subtilisin and IMC domains is provided by a three-dimensional structural model.