Biomedical subjects

Zhiguo Su

Publications and source records attributed to Zhiguo Su.

At least 19 recordsLinked to original sources

PubMed2007

Modeling the interaction of fipronil-related non-competitive antagonists with the GABA beta3-receptor.

A three-dimensional model of the beta3-homopentamer of the gamma-aminobutyric acid (GABA) receptor/chloride ionophore complex was developed by homology modeling using the cyro-electron microscopy structure of nicotinic acetylcholine as a template. Interactions between the beta3-homopentamer and two classes of fipronil-related non-competitive antagonists were investigated using docking studies. The phenyl groups of these compounds were stabilized by strong hydrophobic and hydrophilic interactions with the rings formed by Thr256 and Ala252. Leu253 and Ile255 were involved mainly in hydrophobic contact with the pyrazole moiety. Different substitution at positions 15, 16 and 17 of the pyrazole ring of fipronil resulted in weakening of the hydrogen bonds and hydrophobic interactions between the beta3-receptor and fipronil-related heterocyclic compounds, which maybe the principal cause of the decreased affinities reported in vitro. Moreover, a good correlation between total binding energies calculated by AutoDock and experimentally determined IC(50) values proved our models to be reasonable in predicting the interaction mode of the antagonist with the GABA beta3-receptor.

Cryoelectron Microscopy↗

PubMed2006

One-step separation and purification of 3,4-dihydroxyphenyllactic acid, salvianolic acid B and protocatechualdehyde from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography.

Three kinds of polyphenols of Salvia miltiorrhiza Bunge, 3,4-dihydroxyphenyllactic acid, salvianolic acid B and protocatechualdehyde, were separated and purified in one step with solvent system n-hexane-ethyl acetate-methanol-acetic acid-water (1:6:1.5:1.5:8) by high-speed counter-current chromatography. Acetic acid was successfully used to increase the partition of high polar target compounds in organic phase to modify partition coefficient value. 3,4-Dihydroxyphenyllactic acid, salvianolic acid B and protocatechualdehyde were purified from 100mg water extracted crude sample of Salvia miltiorrhiza Bunge at purity of 97.6%, 94.2% and 98.2% and at yield of 98.6%, 73.6% and 90.2%. High-speed counter-current chromatography together with organic/aqueous solvent system supplied an efficient method to purify water-soluble compounds directly from crude samples of traditional Chinese medicines.

Benzaldehydes↗

PubMed2006

Molecular characterization of recombinant Hepatitis B surface antigen from Chinese hamster ovary and Hansenula polymorpha cells by high-performance size exclusion chromatography and multi-angle laser light scattering.

The molecular weight and size of recombinant Hepatitis B surface antigen (HBsAg) derived from Chinese hamster ovary (CHO) and the Hansenula polymorph have been characterized by high-performance size exclusion chromatography with multi-angle laser light scattering (HPSEC-MALLS). The average molecular weight of CHO-derived HBsAg particle (CHO-rHBsAg) (4921 kDa) was higher than that of H. polymorpha yeast strain (Hans-rHBsAg) (3010 kDa). The size of CHO-rHBsAg (22.1 nm) is nearly the same as that of native HBsAg compared to 18.1 nm for Hans-rHBsAg. The average monomer numbers were found to be 155 for CHO-rHBsAg and 86 for Hans-rHBsAg, respectively. The data obtained support the assumption that the higher immunogenicity of CHO-derived HBsAg is related to its more favorable macromolecular assembly structure.

Animals↗

PubMed2006

Modification of poly(glycidyl methacrylate-divinylbenzene) porous microspheres with polyethylene glycol and their adsorption property of protein.

Rigid porous poly(glycidyl methacrylate-divinylbenzene) (P(GMA-DVB)) microspheres were synthesized through suspension polymerization with a mixture of isooctane and 4-methyl-2-pentonal as the porogen. The microspheres were intended to use as column packing materials for protein separation. However, irreversible adsorption of protein was found on the polymer microsphere. To circumvent the problem, polyethylene glycol (PEG) was coupled to the microspheres. The coupling reaction took place between the hydroxyl group of PEG and the epoxy group of the P(GMA-DVB) solid medium in the presence of boron trifluoride. The density of PEG immobilized onto the P(GMA-DVB) can be determined easily by saponification of modified microsphere firstly and then titration of glycerol-PEG. The effect of the cross-linker content of microsphere on the density of PEG immobilization was investigated. Molecular weight of PEG was found to influence the PEG-immobilization density, which subsequently affects the hydrophilicity of the modified P(GMA-DVB). Bovine serum albumin (BSA) and trypsin were used as model proteins to examine the adsorption and desorption properties of the modified P(GMA-DVB) microspheres. The results demonstrated that P(GMA-DVB) porous microsphere with 20% DVB and modified with PEG4000 showed excellent adsorption and desorption properties. Adsorption capacity of BSA on the modified microsphere attained to 51.6 mg/g microsphere, and BSA mass recovery and trypsin activity recovery was up to 97.6% and 98.7%, respectively. The modified microsphere was demonstrated to be a promising hydrophobic interaction chromatography material for purification of protein.

Adsorption↗

PubMed2006

Mass spectrometric analysis of enzymatic digestion of denatured collagen for identification of collagen type.

Collagen type II and I from bovine were thermally denatured and digested with trypsin. The digest mixture was analyzed with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). Peptides in the digest mixture were identified by mass spectrometry/mass spectrometry (MS/MS) sequencing. The results indicated that the digest mixtures of collagen type II and I contained lots of specific peptides and common peptides. Specific peptides could be used as index for identifying collagen type. Articular cartilage from bovine was pretreated and analyzed with the same method to determine the collagen types. The result indicated that the method developed was effective for identification of collagen types. The research provided a possible approach for collagen identification in particular tissues.

Animals↗

PubMed2006

On-column refolding of consensus interferon at high concentration with guanidine-hydrochloride and polyethylene glycol gradients.

Dilution refolding of consensus interferon (C-IFN) had a limit on final concentration not exceeding 0.1 mg ml(-1) in order to achieve specific activity of 2.2x10(8) U mg(-1). Addition of polyethylene glycol (PEG) only gave a marginal improvement on the specific activity. Hydrophobic interaction chromatography (HIC) was tried but a simple step-wise elution could not refold the protein. Successful refolding was achieved by gradient elution with the decreasing of guanidine-hydrochloride (guanidine-HCl) concentration. The column was packed with a commercially available HIC medium that was designed for protein separation. Polyethylene glycol was found to possess better effect on the column than in the dilution for promotion of correct refolding, especially in gradient mode. A novel dual-gradient strategy, consisting of decreasing guanidine-HCl concentration and increasing PEG concentration, was developed to enhance the refolding yield. Denatured C-IFN was allowed to adsorb and elute from the HIC column through a gradually changed solution environment. Compared with dilution refolding, the gradient HIC process, in the presence of PEG, gave about 2.6-folds of increase in specific activity, 30% increase in soluble protein recovery. Partial purification was also achieved simultaneously.

Chromatography, Liquid↗

PubMed2006

Size characterization of green fluorescent protein inclusion bodies in E. coli using asymmetrical flow field-flow fractionation-multi-angle light scattering.

The goal of this study was to investigate the applicability of asymmetrical flow field-flow fractionation-multi-angle light scattering (AsFlFFF-MALS) for size analysis of green fluorescent protein inclusion bodies (GFPIBs). The size distributions of GFPIBs prepared by various culture conditions were determined. For GFPIBs prepared at 37 degrees C the peak maximum hydrodynamic diameter (d(H)) first increased and then decreased with the increase of the induction times in the presence of 0.1 and 2 mM isopropyl-beta-D-thiogalactoside (IPTG). For GFPIBs prepared at 30 degrees C the peak maximum d(H) was constant at about 700 nm irrespectively of the induction times and IPTG concentrations.

Dose-Response Relationship, Drug↗

PubMed2005

Ion-exchange chromatography of hepatitis B virus surface antigen from a recombinant Chinese hamster ovary cell line.

About 10% of the Chinese population are chronic carriers of hepatitis B virus (HBV). Thus, the development of a highly efficient process for the preparation of a vaccine based on a recombinant hepatitis B surface antigen (HBsAg) is very important to the Chinese national immunization program. To this end, the ion exchange chromatography recovery of CHO-HBsAg from a recombinant Chinese hamster ovary cell line was shown to increase from about 55 to 80% by the addition of 1% poly(ethylene glycol) (PEG 10,000) to the mobile phase. Furthermore, based on analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the intact glycoprotein form of CHO-HBsAg was completely preserved by the addition of PEG. In the absence of PEG the glycoprotein form of CHO-HBsAg was also spread out into the high salt elution fraction. High-performance size-exclusion chromatography with on-line multiangle-laser-light scattering (HPSEC-MALLS) analysis was performed to monitor the status of the CHO-HBsAg aggregate structure assembly, particle size and molecular weight distribution after each purification step, and the results showed further that the presence of PEG facilitated the separation and recovery of intact glycoprotein form of CHO-HBsAg and promoted their assembly to proper virus-like particles, which are both important features and prerequisites of their immunogenicity.

Animals↗

PubMed2005

Reproducible preparation and effective separation of PEGylated recombinant human granulocyte colony-stimulating factor with novel "PEG-pellet" PEGylation mode and ion-exchange chromatography.

A novel preparation for polyethylene glycol (PEG) derivatives and chromatographic separation procedure of the PEGylated recombinant human granulocyte colony-stimulating factor (rhG-CSF) were designed to evaluate the reproducibility and scalability at large laboratory-scale level. The new "PEG-pellet" PEGylation mode was successfully applied to control the pH fluctuation during the conjugation reaction, a general problem in traditional liquid-phase conjugation mode. Moreover, two consecutive ion-exchange chromatography steps were successfully used to separate and purify the PEGylated rhG-CSF. Cation-exchange chromatography was firstly applied to separate PEGylated rhG-CSF from intact rhG-CSF, followed by anion-exchange chromatography to obtain individual PEG-rhG-CSF species (mono-, di- and tri-PEGylated rhG-CSF) and remove the excess free PEG. Furthermore, the molecular weight of individual PEGylated rhG-CSF was identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and SDS-PAGE, and cell proliferation activity in vitro was detected by MTT assay using NFS-60 cell.

Animals↗

PubMed2005

Novel polyethylene glycol derivative suitable for the preparation of mono-PEGylated protein.

A novel methoxypolyethylene glycol (mPEG) derivative, containing a reactive group of 1-methyl pyridinium toluene-4-sulfonate, was synthesized and characterized. The mPEG derivative was successfully conjugated with two proteins: recombinant human granulocyte-colony stimulating factor (rhG-CSF) and consensus interferon (C-IFN). Homogeneous mono-PEGylated proteins were obtained which were identified by high performance size-exclusion chromatography and MALDI-TOF mass spectrometry. The biological activities of the mono-PEGylated rhG-CSF and the mono-PEGylated C-IFN were maintained at 90% and 88%, respectively.

Chromatography, Gel↗

PubMed2005

Disuccinimidyl suberate cross-linked hemoglobin as a novel red blood cell substitute.

Disuccinimidyl suberate (DSS) intramolecularly cross-linked hemoglobin (Hb) was developed as a novel red blood cell substitute. A multi-angle laser light scattering detector coupled with size exclusion HPLC was applied to determine the molecular weight of the modified Hb. SDS-PAGE was also used as a complement. It was proved that 83.8% of the product was intramolecularly cross-linked Hb with weight-average molecular weights (Mw) of 67.5 kD, 12% was dimeric Hb with Mw of 146.6 kD, and 4.2% was trimeric Hb with Mw of 306.4 kD. The tetramer structure of the cross-linked Hb was stable as shown in size-exclusion chromatography using a mobile phase containing 1 mol/L MgCl2. Analysis by LC-MS demonstrated that the reaction of DSS with Hb mainly took place between the two alpha subunits within a Hb molecule, resulting in stabilization of the tetramer structure. However, the cross-linking was not site-specific. The P50 of the cross-linked Hb decreased from 21.8 mmHg to 14.3 mmHg, and the Hill coefficient decreased from 2.22 to 1.41. Result of isoelectric focusing showed that the pl of DSS cross-linked Hb was in the range of 4.6-5.2, similar to that of serum albumin. The safety of DSS cross-linked Hb was favored by animal tests on rats and guinea pigs. Exchange transfusion experiment with DSS cross-linked Hb using rats as a model indicated no pressor effect or other significant side effects. The characteristics and properties of DSS cross-linked Hb were also compared with that of diaspirin cross-linked Hb reported in the literature.

Animals↗

PubMed2004

Polyethylene glycol increases purification and recovery, alters retention behavior in flow-through chromatography of hemoglobin.

Flow-through chromatography was a method for purification of hemoglobin (Hb) from red cell lysate. The presence of 0-5% polyethylene glycol (PEG) increased the retention time of Hb peak from 15 min to 20 min in flow-through ion-exchange chromatography (IEC) but decreased the retention time from 88 min to 62 min in the hydrophobic interaction chromatography (HIC). However, the purification and the recovery were both increased. For IEC the recovery of hemoglobin increased from 75% to more than 90%, and the purified Hb showed single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and one peak in size-exclusion HPLC. For HIC, the recovery of hemoglobin was improved significantly from 20% to 85% and the removal of lipids was 100%. The bioactivity of hemoglobin was well preserved in these two chromatographic processes. The mechanism for the effect of PEG in these two flow-through chromatographic processes was discussed.

Electrophoresis, Polyacrylamide Gel↗

PubMed2004

Fingerprinting of Salvia miltiorrhiza Bunge by non-aqueous capillary electrophoresis compared with high-speed counter-current chromatography.

The component of the traditional Chinese medicine (TCM) can be influenced by soil, climate, and growth stage, and fingerprint is an important means in its quality control. Our previous studies showed that high-speed counter-current chromatography (HSCCC) was helpful in the development of fingerprint of TCM. Since the HSCCC method is new, it is necessary to compare it with conventional ones, such as high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC) and high-performance capillary electrophoresis (HPCE). Comparison with HPLC was conducted in our previous study. In this study, HSCCC was compared with non-aqueous capillary electrophoresis (NACE). With NACE, seven stable components were separated within 55 min, respectively, from three crude samples of Salvia miltiorrhiza Bunge from different growth locations. In HSCCC separation, 12 components were separated, respectively, with good correspondence and precision within 13 h. Both NACE and HSCCC were effective in showing whole concentration distribution of all kinds of constituents. Principles of these two methods were very different, which led to different elution sequences and relative contents of peaks. HSCCC showed better performance in analysis of tanshinones, which made its fingerprint containing more chemical information than that of NACE. It was further proven that HSCCC could be a feasible and cost-effective method in the development of the fingerprint of TCM.

Countercurrent Distribution↗

PubMed2004

Identification of major active constituents in the fingerprint of Salvia miltiorrhiza Bunge developed by high-speed counter-current chromatography.

High-speed counter-current chromatography was applied as a method to develop fingerprinting of Salvia miltiorrhiza Bunge, a popular traditional Chinese medicine, in our previous study. Important active constituents that were directly related to the therapy effect should be identified. Each effluent fraction and standard samples (cryptotanshinone, tanshinoneI and tanshinoneIIA) were analyzed by ultraviolet spectroscopy and liquid chromatography-mass spectroscopy. It was concluded from the UV-Vis spectrograms, retention times in LC analysis and mass spectrograms, that fractions 7, 8 and 11 were respectively cryptotanshinone (Mr 296), tanshinone I (Mr 276) and tanshinone IIA (Mr 294).

Abietanes↗

PubMed2004

Quantitatively investigating monomethoxypolyethylene glycol modification of protein by capillary electrophoresis.

Capillary electrophoresis (CE) was applied to study quantitatively protein modification with succinimidyl succinate-activated monomethoxypolyethylene glycol (MPEG-SS). The heterogeneous distribution of modified proteins and the average modification degree were determined by CE, and the latter met with the results from 2,4,6-trinitrobenzenesulfonic acid (TNBS) spectrometric assay. It was found that the optimal buffer pH for the modification was between pH 7.4 and 8.4, and the modification degree decreased when the modified sample was preserved in high pH solutions. The protein fractions attached with different number of polyethylene glycols (PEGs) were monitored along the process of protein modification. CE was proved to be efficient to evaluate quantitatively several factors of the protein modification, including the modifier/protein molar ratio, the stability of conjugates in different pH environments, and the time course of modification process.

Animals↗

PubMed2004

Comparison of high-speed counter-current chromatography and high-performance liquid chromatography on fingerprinting of Chinese traditional medicine.

Active constituents of Chinese traditional medicine (CTM) are influenced by soils, climates, and growth stages. Therefore, fingerprint is important in quality control of CTM. Our studies showed for the first time that high-speed counter-current chromatography (HSCCC) could be applied in the development of fingerprint of CTM. Twelve components were separated from extracts of three crude samples of Salvia miltiorrhiza Bunge, which were collected from different growth locations. All 12 components of each sample were isolated within 13 h coming with good correspondence by using HSCCC. Relative standard deviation (R.S.D.) values of the retention time in HSCCC separation was less than 3% satisfied the precision requirements of national standards in the fingerprint development. As a new approach, HSCCC was compared to the conventional approach-HPLC in our experiments. The fingerprint developed by HPLC comprised 11 peaks that collected within 45 min. R.S.D. values of retention times of the corresponding peaks in HPLC analysis were very small (maximum 0.66% and average 0.10%). In conclusion, HSCCC was a feasible method to be used in the development of fingerprint of CTM.

Chromatography, High Pressure Liquid↗

PubMed2004

Conformational changes of lysozyme refolding intermediates and implications for aggregation and renaturation.

It is believed that denatured-reduced lysozyme rapidly forms aggregates during refolding process, which is often worked around by operating at low protein concentrations or in the presence of aggregation inhibitors. However, we found that low concentration buffer alone could efficiently suppress aggregation. Based on this finding, stable equilibrium intermediate states of denatured-reduced lysozyme containing eight free SH groups were obtained in the absence of redox reagents in buffer of low concentrations alone at neutral or mildly alkaline pH. Transition in the secondary structure of the intermediate from native-like to beta-sheet was observed by circular dichroism (CD) as conditions were varied. Dynamic light scattering and ANS-binding studies showed that the self-association accompanied the conformational change and the structure rich in beta-sheet was the intermediate state for aggregation, which could form either amyloid protofibril or amorphous aggregates under different conditions as detected by Electron Microscopy. Combining the results obtained from activity analysis, RP-HPLC and CD, we show that the activity recovery was closely related to the conformation of the refolding intermediate, and buffer of very low concentration (e.g. 10mM) alone could efficiently promote correct refolding by maintaining the native-like secondary structure of the intermediate state. This study reveals reasons for lysozyme aggregation and puts new insights into protein and inclusion body refolding.

Amyloid↗

PubMed2004

A novel tetrafunctional reagent for simultaneous intramolecular and intermolecular cross-linking of bovine hemoglobin.

A novel tetrafunctional reagent, alpha,gamma,alpha',gamma'-tetra-succinimidyl-hexanediamide-di-glutamate ester (HDG(OSu)(4)), was successfully synthesized, and a well-defined cross-linked bovine hemoglobin (mainly 128 kDa) was prepared with this reagent. Due to the spatial structure of this cross-linking reagent, the intramolecular and intermolecular cross-linking of bovine hemoglobin was formed simultaneously in one reaction. Although the cross-linked bovine hemoglobin showed a slight decrease in half-saturated O(2) pressure value (P(50), from 28.1 mm Hg to 21.7 mm Hg) and Hill coefficient (from 2.5 to 2), due to the cross-linkage, it still performed well for O(2) delivery.

Animals↗