Shaowu Lv

A trifunctional enzyme with glutathione S-transferase, glutathione peroxidase and superoxide dismutase activity

Superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS, as not one of them can singlehandedly clear all forms of ROS. In order to imitate the synergy of the enzymes, we designed and generated a recombinant protein, which comprises of a Schistosoma japonicum GST (SjGST) and a bifunctional 35-mer peptide with SOD and GPX activities. The engineered protein demonstrated SOD, GPX and GST activities simultaneously. This trifunctional enzyme with SOD, GPX and GST activities is expected to be the best ROS scavenger.

A novel dicyclodextrinyl diselenide compound with glutathione peroxidase activity

A 6A,6A′‐dicyclohexylamine‐6B,6B′‐diselenide‐bis‐β‐cyclodextrin (6‐CySeCD) was designed and synthesized to imitate the antioxidant enzyme glutathione peroxidase (GPX). In this novel GPX model, β‐cyclodextrin provided a hydrophobic environment for substrate binding within its cavity, and a cyclohexylamine group was incorporated into cyclodextrin in proximity to the catalytic selenium in order to increase the stability of the nucleophilic intermediate selenolate. 6‐CySeCD exhibits better GPX activity than 6,6′‐diselenide‐bis‐cyclodextrin (6‐SeCD) and 2‐phenyl‐1,2‐benzoisoselenazol‐3(2H)‐one (Ebselen) in the reduction of H2O2, tert‐butyl hydroperoxide and cumenyl hydroperoxide by glutathione, respectively. A ping‐pong mechanism was observed in steady‐state kinetic studies on 6‐CySeCD‐catalyzed reactions. The enzymatic properties showed that there are two major factors for improving the catalytic efficiency of GPX mimics. First, the substrate‐binding site should match the size and shape of the substrate and second, incorporation of an imido‐group increases the stability of selenolate in the catalytic cycle. More efficient antioxidant ability compared with 6‐SeCD and Ebselen was also seen in the ferrous sulfate/ascorbate‐induced mitochondria damage system, and this implies its prospective therapeutic application.

A DNA sensor based on surface plasmon resonance for apoptosis-associated genes detection.

A practical and simple DNA sensor based on surface plasmon resonance (SPR) had been developed to determine apoptosis-associated genes, bcl-2 and bax. This SPR sensor was designed on the basis of simultaneous multi-wavelength detection. The complementary sequences of bcl-2 or bax oligonucleotide labeled with biotin were used as the probes. Biotin-avidin system was used to immobilize the bio-DNA on the sensor surface. The assembling processes and conditions for the DNA sensor were examined. The SPR sensor could be used to monitor the process of the immobility of the bio-DNA and DNA hybridization in real-time. The determination range of bcl-2 and bax oligonucleotide (20 bases) were 50-400 ng/mL. The determination range of polymerase chain reaction (PCR) product of bcl-2 (405 bases) was 5-60 ng/mL and PCR product of bax (538 bases) was 5-40 ng/mL. The stability, reversibility and specificity of the DNA sensor were also investigated. It was found from the experiment that the sensor could be applied for a quite long time (about 90 times). The relative standard deviation (R.S.D.) for determination oligonucleotides and PCR products of bcl-2 were 1.2 and 1.3%, respectively. The interference of noncomplementary DNA sequence with the determination of DNA was examined and it was found that noncomplementary 20-mer and 21-mer DNA (p53 and p21) do not affect the determination of bcl-2 or bax. This device could be used to study apoptosis and signal transduction routine genes. The sensor was shown to be of simplicity, sensitivity, selectivity, rapid response and cost effectiveness.

Fluorescence turn-on assay for glutathione reductase activity based on a conjugated polyelectrolyte with multiple carboxylate groups

In this work, an anionic conjugated polyelectrolyte (CPE) that features four carboxylate groups on each repeat unit, PPE-(COOK)4, has been designed and synthesized by integrating the two synthetic strategies for conjugated poly(phenylene ethynylene) and dendritic polyamidoamine. Through the installation of multiple functional groups, the proposed CPE has been greatly improved in many aspects, such as the good water solubility, slight aggregation, and relatively high quantum yield. Thanks to the close resemblance in chemical structures between the polymers pendant groups and EDTA, the resulting PPE-(COOK)4 has also been found to show stronger complexation with metal ions, and has further been demonstrated to be an excellent fluorescent probe for Cu2+ with both high sensitivity and remarkable specificity. Moreover, by taking advantage of the distinct fluorescence recovery rates of PPE-(COOK)4/Cu2+ caused by GSH and by GSSG, a novel fluorescence turn-on assay for glutathione reductase (GR) has successfully been developed. With a minimum detectable enzyme concentration of 0.2 mU/mL, this newly proposed GR activity assay is highly sensitive and robust as compared to most spectrophotometric and fluorescent methods.

A novel selenium-containing glutathione transferase zeta1-1, the activity of which surpasses the level of some native glutathione peroxidases

Glutathione peroxidase (GPX) is a critical antioxidant selenoenzyme in organisms that protects cells against oxidative damage by catalyzing the reduction of hydroperoxides by glutathione (GSH). Thus, some GPX mimics have been generated because of their potential therapeutic value. The generation of a semisynthetic selenoenzyme with peroxidase activity, which matches the catalytic efficiencies of naturally evolved GPX, has been a great challenge. Previously, we semisynthesized a GPX mimetic with high catalytic efficiency using a rat theta class glutathione transferase (rGST T2-2) as a scaffold, in which the highly specific GSH-binding site is adjacent to an active site serine residue that can be chemically modified to selenocysteine (Sec). In this study, we have taken advantage of a new scaffold, hGSTZ1-1, in which there are two serine residues in the active site, to achieve both high thiol selectivity and highly catalytic efficiency. The GPX activity of Se-hGSTZ1-1 is about 1.5 times that of rabbit liver GPX, indicating that the selenium content at the active site plays an important role in enhancement of catalytic performance. Kinetic studies revealed that the catalytic mechanism of Se-hGSTZ1-1 belong in a ping-pong mechanism similar to that of the natural GPX.

Human catalytic antibody Se-scFv-B3 with high glutathione peroxidase activity.

In order to generate catalytic antibodies with glutathione peroxidase (GPX) activity, we prepared GSH‐S‐2,4‐dinitrophenyl t‐butyl ester (GSH‐S‐DNPBu) as target antigen. Three clones (A11, B3, and D5) that bound specifically to the antigen were selected from the phage display antibody library (human synthetic VH + VL single‐chain Fv fragment (scFv) library). Analysis of PCR products using gel electrophoresis and sequencing showed that only clone B3 beared intact scFv‐encoding gene, which was cloned into the expression vector pPELB and expressed as soluble form (scFv‐B3) in Escherichia coli Rosetta. The scFv‐B3 was purified by Ni2+‐immobilized metal affinity chromatography (IMAC). The yield of purified proteins was about 2.0–3.0 mg of proteins from 1 L culture. After the active site serines of scFv‐B3 were converted into selenocysteines (Secs) with the chemical modification method, we obtained the human catalytic antibody (Se‐scFv‐B3) with GPX activity of 1288 U/µmol. Copyright

A novel 65-mer peptide imitates the synergism of superoxide dismutase and glutathione peroxidase.

Reactive oxygen species (ROS) are involved in cell growth, differentiation, and death. Excessive amounts of ROS (e.g., O(2)(-), H(2)O(2), and HO) play a role in aging as well as in many human diseases. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) are critical antioxidant enzymes in living organisms. SOD catalyzes the dismutation of O(2)(-) to H(2)O(2), and GPx catalyzes the reduction of H(2)O(2) and other harmful peroxides by glutathione (GSH). They not only function in catalytic processes but also protect each other, resulting in more efficient removal of ROS, protection of cells against injury, and maintenance of the normal metabolism of ROS. To imitate the synergism of SOD and GPx, a 65-mer peptide (65P), containing sequences that form the domains of the active center of SOD and the catalytic triad of GPx upon the incorporation of some metals, was designed on the basis of native enzyme structural models; 65P was expressed in the cysteine auxotrophic expression system to obtain Se-65P. Se-65P was converted into Se-CuZn-65P by incorporating Cu(2+) and Zn(2+). Se-CuZn-65P exhibited high SOD and GPx activities because it has a delicate dual-activity center. The synergism of the enzyme mimic was evaluated by using an in vitro model and a xanthine/xanthine oxidase/Fe(2+)-induced mitochondrial damage model system. We anticipate that the peptide enzyme mimic with synergism is promising for the treatment of human diseases and has potential applications in medicine as a potent antioxidant.

A new human catalytic antibody Se-scFv-2D8 and its selenium-containing single domains with high GPX activity

Glutathione peroxidase (GPX) is a well‐known antioxidant selenoenzyme, which can catalyze the reduction of a variety of hydroperoxides and consequently protect cells and other biological tissues against oxidative damage. Many attempts have been made to mimic its function, and a human catalytic antibody Se‐scFv‐B3 with GPX activity has been prepared in our previous study. This time, a new clone 2D8 that bound specifically to the glutathione analog GSH‐S‐DNPBu was selected again by using the technology of phage display antibody library, and then scFv‐2D8 was successfully expressed in soluble form and purified using Ni2+‐immobilized metal affinity chromatography. After being converted into selenium‐containing scFv by chemically modification, it showed higher GPX activity than previous abzyme Se‐scFv‐B3. The heavy chain variable fragment of scFv‐2D8 was also prepared and converted into selenium‐containing protein using the same method. This selenium‐containing single‐domain antibody showed some GPX activity and, to the best of our knowledge, is the first human single‐domain abzyme with GPX activity, which lays a foundation for preparing GPX abzyme with human origin, lower molecular weight and higher activity. Copyright

Protection of epidermal cells against UVB injury by the antioxidant selenium-containing single-chain Fv catalytic antibody

The antioxidant effect of selenium-containing single-chain Fv catalytic antibody (Se-scFv2F3), a new mimic of glutathione peroxidase, was confirmed using a model system in which cultured rat skin epidermal cells were injured by ultraviolet B (UVB). The cell damage was characterized in terms of lipid peroxidation of the cells, cell viability, and cell membrane integrity. The injury effects of UVB and protection effects of Se-scFv2F3 on the cells were studied using the model system. UVB can damage the cells severely. Upon precultivation of the cells with 0.4U/ml Se-scFv2F3, however, the damage was significantly reduced as shown by the increase in cell viability, the decrease in the malondialdehyde and hydrogen peroxide levels, and the normalization of lactate dehydrogenase activity. In addition, a novel finding that Se-scFv2F3 can stimulate cultured epidermal cells to proliferate under certain conditions was observed.

Development of particle swarm optimization–support vector regression (PSO-SVR) coupled with microwave plasma torch–atomic emission spectrometry for quality control of ginsengs

As people have become more focused on their own health, the role of ginseng for medical uses has begun to receive substantial interest. However, the quality control of ginseng remains in question because different species vary considerably in this respect. In this paper, particle swarm optimization–support vector regression combined with microwave plasma torch–atomic emission spectrometry (MPT‐AES) was used, for the first time, for quality control of ginseng. To build calibration models, quantitative determination of target element concentrations in ginseng samples was conducted by MPT‐AES because ginseng quality was closely related to the place of origin and can thus be judged by the elemental composition. Characteristic spectral lines were extracted via principal component analysis to reduce the computational effort and improve the representativeness of the input variables. Two heuristic algorithms, particle swarm optimization and a genetic algorithm, were selected to optimize the parameters (eg, c, g, and ε) that were extremely significant in the construction of the support vector regression (SVR) models. Another linear regression approach, partial least squares regression (PLSR), was also used and compared. The comparisons were based on evaluation indexes, namely, the root mean square error and the squared correlation coefficient (R2). A significant difference between SVR and PLSR showed that SVR outperformed PLSR in such a multivariate regression problem. The acquired results showed that particle swarm optimization was slightly better than a genetic algorithm. In conclusion, the proposed MPT‐AES combined with particle swarm optimization–support vector regression is appropriate for quantitative elemental analysis and further application in the quality control of ginseng.