THERMODYNAMIC STUDY OF ADSORPTION OF PHENOLIC COMPOUNDS FROM AQUEOUS SOLUTION BY A WATER-COMPATIBLE HYPERCROSSLINKED POLYMERIC ADSORBENT

Equilibrium data for the adsorption of phenolic compounds, i.e., phenol, p-cresol, p-chlorophenol and p-nitrophenol from aqueous solutions by a water-compatible hypercrosslinked polymeric adsorbent (NJ-8) within temperature range of 283-323 K were obtained and correlated with a Freundlich-type of isotherm equation, so that equilibrium constants KF and n were obtained. The capacities of equilibrium adsorption for all the four phenolic compounds on the NJ-8 from aqueous solutions are around 2 times as high as those of Amberlite XAD-4, which may be attributed to the unusual micropore structure and the partial polarity on the network. The values of the enthalpy (always negative) are indicative of an exothermic process, which manifests the adsorption of all the four phenolic compounds on the two polymeric adsorbents to be a process of physical adsorption. The negative values of free energy change show that the solute is more concentrated on the adsorbent than in the bulk solution. The absolute free energy values of adsorption for NJ-8 are always higher than those for Amberlite XAD-4, which indicates that phenolic compounds are preferentially adsorbed on NJ-8. The negative values of the adsorption entropy are consistent with the restricted mobilities of adsorbed molecules of phenolic compounds as compared with the molecules in solution. The adsorption entropy values of phenolic compounds for NJ-8 are lower than those for Amberlite XAD-4, which means the micropores of NJ-8 require more orderly arranged adsorbate.     

INTRAPARTICLE DIFFUSION OF RARE EARTHS IN POROUS CATION EXCHANGERS

Experiments for determining cerium isotope ion exchange rates with macrop-orous resins Amberlyst 15, D001 and XN1010 are discribed. The kinetics of the isotope ion exchange reaction has been examined by a simple theoretical equation of intraparticle effective diffusivity De in a porous ion exchanger. The ion exchange proceeds by diffusion within the macropores and the solid phase of the resin. De of cerium was affected by the concentration of the bulk solution C and was separated into a macropore diffusivity Dp and a solid phase diffusivity Dg by the equation. The diffusion coefficients of the exchanging ion are shown to have the values in the macropores comparable with those in the bulk solution and to have the values in the solid phase comparable with those in gel resin with the same crosslinkage as the resins used for the experiments.     

FKBP23 in ER binds to BiP specifically

FKBP23 was found in mouse endoplasmic reticulum (ER) in 1998. It consists of an N-terminal peptidyl-prolyl cis-trans isomerase (PPIase) domain and a C-terminal domain with Ca2+ binding sites. The fusion protein of mouse FKBP23 and glutathione S-transferase (GST), GST-FKBP23, and the fusion protein of BiP, a member of heat shock protein 70 (Hsp 70) in ER, and GST, GST-BiP, were subcloned in E. coli, expressed and purified. The fusion proteins were restrictively digested by Factor Xa (FaXa) to obtain the free cloned proteins FKBP23 and BiP. With the assay of adsorption of free FKBP23 or BiP with GST-BiP or GST-FKBP23 attached to the Glutathione-Sepharose 4B, the adsorbed FKBP23 or BiP could be detected by Immunoblot. It means that FKBP23 binds to BiP. Furthermore, BiP in leukocyte ER-extract can be adsorbed with GST-FKBP23 attached to the glutathione-Sepharose 4B. It shows that FKBP23 binds to natural BiP in ER, too. These experiments show that a PPIase binds to a molecular chaperone of the Hsp70 family.     

Expression,Purification and Spectral Characterization of p21Waf1/Cip1

p21^wafl/cipl, best known as a broad-specificity inhibitor of cyclin/cyclin-dependent kinase complexes, can interact with various target proteins, and this ability relies on its structural plasticity. Therefore, studies on the structural properties of p21 are very important to understand its structure-function relationship. However, detailed studies on its secondary structure and biophysical propertics have been comparatively sparse. A human p21 gene was cloned into the temperature expression vector pBV220 and transformed into Escherichia coli strain JM109. Recombinant protein was expressed as a non-fusion protein and purified by gel filtration and anion exchange chromatography. The purified protein was verified by Western blot and the functional activity was recognized by pull-down assay. Furthermore, circular dichroism, fluorescence spectroscopy, and fluorescence quenching methods were used to characterize the conformational properties of the purified protein. The results indicate that it was largely unstructured under the native solution conditions, and its tryptophan residues were exposed and located in a positively charged microenvironment. This study lays a good foundation for further study of p21 binding to its different partners.     

STRUCTURE, MOLECULAR WEIGHT AND BIOACTIVITIES OF （1→3）-β-D-GLUCANS AND ITS SULFATED DERIVATIVES FROM FOUR KINDS OF LENTINUS EDODES

Abstract Lentinan samples, (1→3)-β-D-glucans containing 4.6-15.2 wt% proteins, coded as L-I1. L-I2. L-I3 and L-I4 (L-I)were isolated from four kinds of Lentinus edodes. These glucans were treated with acetone to remove the protein in order to obtain free protein glucans coded as LNP-I1. LNP-I2, LNP-I3 and LNP-I4 (LNP-I). The free-protein polysaccharides were sulfated to give derivatives (S-LNP-I) with degree of substitution (DS) from 0.4-0.8. The structural features and weight- average molecular weight (Mw) of the samples were investigated by using infrared spectroscopy, elemental analysis,^13C-NMR, size exclusion chromatography combined with laser light scattering (SEC-LLS) and viscometry. The effects of structure and conformation of the polysaccharides on antitumor activities were assayed in vivo (Sarcoma 180 solid tumors)and in vitro (Sarcoma 180, HL-60, MCF-7 and Vero tumors). The results indicated that the predominant species of the samples L-I and LNP-I in 0.2 mol/L NaCl aqueous solution existed as triple-helical chains with high rigidity and in dimethyl sulfoxide (DMSO) as single-flexible chains. Interestingly, the antitumor activities of LNP-I are lower than those of the native glucans (L-I), whereas their sulfated derivatives have higher inhibition ratio against Sarcoma 180 than LNP-I. The results reveal that the binding of protein, sulfated modification and the triple helix conformation are important factors in the enhancement of the antitumor activities of polysaccharides on the whole.     

PROPERTIES AND THERMODYNAMICS OF ADSORPTION OF BENZOIC ACID ONTO XAD-4 AND A WATER-COMPATIBLE HYPERCROSSLINKED ADSORBENT

The adsorption behavior of benzoic acid onto a water-compatible hypercrosslinked polymeric adsorbent NJ-8 wascompared with that onto macroporous Amberlite XAN-4. This paper focuses on the static equilibrium adsorption behaviors,the adsorption thermodynamics and the column dynamic adsorption profiles. Five isotherm models are used to fit the results.This shows that the Freundlich equation can give a perfect fit. The specific surface area of NJ-8 is about as high as that ofAmberlite XAD-4, but the adsorbing capacity for benzoic acid on NJ-8 is about 14.9%-64.8% higher than that on AmberliteXAD-4, which is attributed to its microporous mechanism and partial polarity. The negative values of the adsorptionenthalpy are indicative of an exothermic process. Both enthalpy and free energy changes of adsorption manifest a physicalsorption process. The negative values of the adsorption entropy indicate that adsorption is well consistent with the restrictedmobilities and the configurations of the adsorbed molecules on the surface of the studied adsorbents with superficialheterogeneity. Both adsorbents were used in mini-column experiments to demonstrate the higher breakthrough adsorbing capacity of the hypercrosslinked polymeric adsorbent NJ-8 to benzoic acid, as compared with that of Amberlite XAD-4.     

Conformer Pair Contributions to Optical Rotations in a Series of Chiral Linear Aliphatic Alcohols

The chain length effect of four chiral aliphatic alcohols,(S)-2-butanol,(S)-2-pentanol,(S)-2-hexanol and (S)-2-heptanol,on their specific optical rotations(OR)was studied experimentally and theoretically via quantum theory.Many conformations of each chiral alcohol exist as conformer pairs in solution.The OR sum from these pairs of conformers has much smaller contributions to OR values than that contributed by the most stable conformation. These four alcohols'OR values were also investigated using the matrix model,which employs each substituent's comprehensive mass,radii,electronegativity and symmetry number as the elements in the matrix.These are all particle properties.This matrix determinant is proportional to its OR values within a closely related structural series of chiral compounds.The experimental OR values and the matrix determinants of these four alcohols were compared with the predicted OR values obtained from quantum theory wave functions.The ORs predicted by the matrix method, which is based on particle function statistics,agreed with the results from quantum theory.The agreement between OR predictions by the matrix method and DFT calculations illustrates the wave-particle duality of polarized light that is operating in these predictions.     

Molecular Dynamics Simulations of the Interactions between Konjac Glucomannan and Carrageenan

The interactions between konjac glucomannan and carrageenan were studied with the method of molecular dynamics simulation. Part representative structure segments of KGM and two unit structures of κ-carrageenan （Fig. 2） were used as mode, and the force-field was AMBER2. The stability and sites of konjac glucomannan/carrageenan interactions in water were researched at 373 K with the following results： the potential energy （EPOT） of the mixed gel was dropped, while those of single-konjac glucomannan gel and single carrageenan were increased. The surface area （SA） of KGM in the mixed system was decreased to 1002.2A^°^2, and that of carrageenan to 800.9 A^°^2. The variations of two parameters showed that the stability of compound gel konjac glucomannan/carrageenan was improved, which is consistent with the previous studies. The sites of interactions in the mixed gel were the -OH groups on C（2）, C（4） and C（6）, the acetyl group in KGM mannose, and the -OH group on C（6） in carrageenan. The hydrogen bond was formed directly or indirectly by the bridge of waters.     

Purification and Characterization of Protein Tyrosine Phosphatase MEG1 and Preparation of Anti-PTPMEG1 Antibody

<正>PTPMEG1 is an intracellular protein tyrosine phosphatase(PTP),which contains FERM and PDZ domains. This study focuses our attention on the expression,purification and characterization of catalytic domain of PTPMEG1(△MEG1) and preparation of its polyclonal antibody.A cDNA fragment encoding△MEG1 protein(amino acid residues 643—926) was amplified by PCR and then cloned into the pT7-7 vector.Both soluble and insoluble recombinant△MEG1 proteins were observed after induction by IPTG.Soluble△MEG1 was purified via two chromatographic steps,and the purified enzyme was characterized.With para-nitrophenylphosphate(pNPP) as a substrate,△MEG1 exhibited typical enzymatic characteristics of classic PTPs and classical Michaelis-Menten kinetics.Insoluble△MEG1,which was mainly distributed in the inclusion body of E.coli cells extracts,was purified by preparative electrophoresis gel for the preparation of the polyclonal antibody.A rabbit was immunized with△MEG1 purified by preparative electrophoresis to generate anti-△MEG1 antibody.Anti-serum was collected on 28th day after initial injection and purified via affinity chromatography.The purified polyconal antibody displayed a satisfactory titer and sensitivity.     

Proteomics Dissection of Cold Responsive Proteins Based on PEG Fractionation in Arabidopsis

Proteome profiling was performed on Arabidopsis plant exposed to cold stress at 4 ℃ for 24 h in an attempt to explore the mechanisms of plant climate adaptation.The polyethylene glycol（PEG） fractionation protocol developed in this lab was used to identify as many differentially expressed low-abundance proteins as possible.In comparison with those of the biological controls,67 protein spots with at least two-fold difference in expression were identified for the plant exposed to cold temperatures; and from these spots,50 proteins were successfully identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry（MALDI-TOF MS）.Bioinformatics studies on these proteins show that 57.8％ of these proteins were localized in the chloroplast.Of these proteins,8 ones have functions in photosynthesis,including glycine hydroxymethyltransferase,Rubisco large subunit,Rubisco activase,PSBO2,fructose-1,6-bisphosphate aldolase,NADP-dependent malate dehydrogenase,sedoheptulose bisphosphatase and photosystem Ⅱ reaction center PsbP family protein,suggesting that photosynthesis is greatly affected by cold stress.The identified proteins were validated by quantitative real-time polymerase chain reaction（qPCR）.Taken together,our results suggest that the chloroplast might also act as a cold stress sensor and that photosynthesis-related proteins may play important roles in cold acclimation for Arabidopsis.     

Affinity purification of Schistosoma japonicum glutathione-S-transferase and its site-directed mutants with glutathione affinity chromatography and immobilized metal affinity chromatography.

A C-terminally polyhistidine-tagged protein of Schistosoma japonicum glutathione-S-transferase, named as SjGST/His, and its Cys85-->Ser, Cys138-->Ser, and Cys178-->Ser site-directed mutants were prepared and highly expressed in Escherichia coli. Both immobilized metal affinity chromatography (IMAC) and glutathione (GSH) affinity chromatography were used to purify these four enzymes. All of them were purified with equal efficiency by Ni2+-chelated nitrilotriacetic acid agarose gel, but not by GSH Sepharose 4B gel. The protein amounts of wild-type and Cys85-->Ser enzymes purified by the latter gel were three to seven-fold greater than those of the other two enzymes purified by the same gel, while their specific activities were two-fold lower, presumably because of the occurrence of noncovalent aggregation. Both purification methods yielded highly pure enzymes, while there were minor amounts of inter- and intra-disulfide forms in the IMAC purified enzymes except for the Cys85-->Ser mutant. Addition of dithiothreitol to GSH-affinity purified enzymes shifted all of their mass spectra of matrix-assisted laser desorption/ionization-time of flight mass spectrometry toward low molecular-mass regions, while addition of GSH to IMAC purified enzymes shifted the spectra toward high molecular-mass regions. The shift values of wild-type enzyme were larger than those of the three mutants, indicating that the Cys85, Cys138, and Cys178 residues were S-thiolated by GSH during the GSH-affinity purification. This result was confirmed by isoelectric focusing. These findings suggest that IMAC is more efficient than the conventional GSH-affinity system for the purification of SjGST/His enzyme, especially for its mutants and fusion proteins.     

Purification and partial characterization of phospholipase A2 isoforms from human placenta.

Five isoforms of the human placental phospholipase A2 were identified and purified to near homogeneity. The purification of these enzymes involved gel permeation, ion-exchange and affinity chromatography. The apparent relative molecular mass of these proteins is 70,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These enzymes have pH optima of 7 and 8. Two-dimensional gel electrophoresis of these enzymes revealed distinct pH optima for each of the isoforms with values ranging from 4.0 to 6.5. Three of the isoforms require calcium for activity whereas the other two forms exhibit 50% of their maximum activity without the presence of calcium.     

Prokaryotic Expression and Polyclonal Antibody Preparation of PRL3

Phosphatase of regenerating liver 3(PRL3),which belongs to the superfamily of protein tyrosine phosphatases(PTPs),represents a group of low molecular weight PTPs that participate in tumorigenesis and metastasis processes.Presented here are the results of cloning,prokaryotic expression,purification,and polyclonal antibody preparation of PRL3.To obtain a specific polyclonal antibody against PRL3,the authors have prepared GST-PRL3 to immunize rabbits and purify an anti-PRL3 polyclonal antibody by negative selection affinity columns.Western blot analysis shows that the anti-PRL3 polyclonal antibody has a specific binding ability with PRL3 protein.The anti-PRL3 polyclonal antibody provides a good tool to further study the function of PRL3.     

Estimation of isoelectric points of human plasma proteins employing capillary isoelectric focusing and peptide isoelectric point markers

Synthetic UV-detectable peptide pI markers were used to estimate isoelectric point (pI) values of proteins separated by capillary isoelectric focusing (CIEF) followed by cathodic mobilization in the absence of denaturing agents. The pI calculation and quantitative analysis of purified proteins showed the feasibility of these peptides as pI markers and internal standards in CIEF separation of proteins. Estimation of pI values of major proteins in human plasma was performed using the peptide pI markers, and the values were compared with those previously obtained by gel isoelectric focusing (IEF). Sera of immunoglobulin G (IgG) myeloma patients, which showed characteristic peaks of myeloma IgG in their CIEF patterns, were also subjected to the analysis and the pI values of the myeloma proteins have been estimated.     

Mapping and identification of protein-protein interactions by two-dimensional far-Western immunoblotting

Studies of protein-protein interactions have proved to be a useful approach to link proteins of unknown function to known cellular processes. In this study we have combined several existing methods to attempt the comprehensive identification of substrates for poorly characterized human protein tyrosine phosphatases (PTPs). We took advantage of so-called "substrate trapping" mutants, a procedure originally described by Flint et al. (Proc. Natl. Acad. Sci. USA 1997, 94, 1680-1685) to identify binding partners of cloned PTPs. This procedure was adapted to a proteome-wide approach to probe for candidate substrates in cellular extracts that were separated by two-dimensional (2-D) gel electrophoresis and blotted onto membranes. Protein-protein interactions were revealed by far-Western immunoblotting and positive binding proteins were subsequently identified from silver-stained gels using tandem mass spectrometry. With this method we were able to identify possible substrates for PTPs without using any radio-labeled cDNA or protein probes and showed that they corresponded to tyrosine phosphorylated proteins. We believe that this method could be generally applied to identify possible protein-protein interactions.     

Inhibitors of Protein Tyrosine Phosphatases: Next‐Generation Drugs?

The protein tyrosine phosphatases (PTPs) constitute a family of closely related key regulatory enzymes that dephosphorylate phosphotyrosine residues in their protein substrates. Malfunctions in PTP activity are linked to various diseases, ranging from cancer to neurological disorders and diabetes. Consequently, PTPs have emerged as promising targets for therapeutic intervention in recent years. In this review, general aspects of PTPs and the development of small-molecule inhibitors of PTPs by both academic research groups and pharmaceutical companies are discussed. Different strategies have been successfully applied to identify potent and selective inhibitors. These studies constitute the basis for the future development of PTP inhibitors as drugs.     

Determination of the Molecular Weight of Extracellular Ribonuclease Isoenzymes from Aspergillus Niger in Crude Extracts by Thin Layer Gel Filtration and Polyacrylamide Gel Electrophoresis

Abstract

The extracellular ribonucleases from Asperqillus niger culture medium were fractionated according to their molecular weight by thin layer gel filtration through Sephadex G100 superfine and the enzyme activity was detected by a standard staining technique on a replica print paper. Another replica paper was laid onto the top of a polyacrylamide gel and the absorbed proteins were separated by electrophoresis. By comparing the electrophoretic pattern with that of a control not subjected to gel filtration, the molecular weight of each isoenzyme in the crude extract could be determined. Gel electrophoresis however, is only used to establish the correspondence between the original electrophoretic pattern of the isoenzymes in the crude preparation and that detected on the replica print paper taken after the thin layer gel filtration run. There was good agreement between the values obtained for the crude and purified enzymes.     

Size-based separations of proteins by capillary electrophoresis using linear polyacrylamide as a sieving medium: model studies and analysis of cider proteins

Electrophoretic conditions to separate sodium dodecyl sulfate (SDS)-protein complexes according to their relative molecular mass by capillary electrophoresis (CE) using linear polyacrylamide as a sieving matrix were examined. Five purified proteins with relative molecular masses between 14 400 and 66 200 Da were separated on a coated fused-silica capillary with an internal diameter of 100 microm and an effective length of 24 cm (total length, 32.5 cm). Benzoic acid was added to the solution of purified proteins as internal standard; beta-mercaptoethanol was also added as reducing agent. The running buffer composition was 0.05 M tris(hydroxymethyl)aminomethane (Tris), 0.035 M aspartic acid, 0.1% m/v SDS, 4% m/v acrylamide, the resulting pH being 8.0. The applied voltage was 7 kV (reversed voltage polarity) in order to avoid high current intensities. Under optimized conditions, the five proteins were separated in less than 15 min, with a % relative standard deviation (RSD) between 0.2 and 0.4 for migration times in the same day. Good efficiency (values between 150 000 and 40 000 N/m) and resolution (values between 2 and 2.8) were obtained. The inverse of relative migration times was found to correlate with the logarithm of their relative molecular mass. Finally, cider proteins were analyzed and their relative molecular masses were determined. These results were compared with those obtained by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).     

Purification and partial characterization of the pancreatic proteolytic enzymes trypsin, chymotrypsin, and elastase from the chicken.

The purpose of this work was to isolate, purify and partially sequence trypsin, chymotrypsin and elastase from the chicken pancreas. The extraction of the pancreatic zymogens with 0.5 M CaCl2 at pH 7.5 for 9 h appeared to be most effective in obtaining maximum recovery of the three enzymes. The sequential Cucurbita maxima trypsin inhibitor I/bovine pancreas trypsin inhibitor/soybean trypsin inhibitor affinity chromatography gave the best result for the isolation of trypsin, chymotrypsin and elastase, respectively, from the same extract. For each proteinase, multiple form of enzymatic activity could be observed after gel electrophoresis and each form was further purified on an ion-exchange column. The N-terminal amino acid sequence of trypsin and chymotrypsin showed homologies with the bovine enzymes whereas elastase showed homologies with the porcine enzyme. The molecular mass of trypsin, chymotrypsin and elastase were estimated to be 23,500, 25,700 and 25,000, respectively, which are values close to those in mammalian species. Although some kinetic constants (Km and k(cat)/Km) appeared different from those observed in other species, the pH dependent enzymatic activities were similar to those reported in other animal species.     

Characterization of aldose reductase and aldehyde reductase from the medulla of rat kidney

Aldose reductase and aldehyde reductase from the medulla of the rat kidney have been purified to homogeneity by using affinity chromatography, gel filtration and chromatofocusing. The molecular weights of aldose reductase and aldehyde reductase by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were found to be 37000 and 39000, respectively. The isoelectric points of aldose reductase and aldehyde reductase were found to be 5.4 and 6.2 by chromatofocusing, respectively. The major differences of amino acid compositions between both enzymes were found in serine, alanine and aspartic acid. Substrate specificity studies showed that aldose reductase utilized aldo-sugars such as D-glucose and D-galactose, but aldehyde reductase did not use them. The Km values of aldose reductase for various substrates were lower than those of aldehyde reductase. Aldose reductase utilized both reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduced nicotinamide adenine dinucleotide (NADH) as coenzymes, whereas aldehyde reductase utilized only NADPH. The presence of the sulfate ion resulted in a dramatic activation of aldose reductase whereas it did not affect aldehyde reductase activity. These enzymes were strongly inhibited by the known aldose reductase inhibitors. However, aldose reductase was more susceptible than aldehyde reductase to inhibition by the aldose reductase inhibitors.