TSK G3000SW Column Research Articles

Application of high-performance size-exclusion liquid chromatography to the study of copper speciation in waters extracted from sewage sludge treated soils

A simple linear relationship has been established between the ionic strength and electrical conductivity of soil solutions. This relationship allows the preparation of an eluent for high-performance liquid chromatography which resembles the soil solution in terms of pH and ionic strength without requiring a complete ionic analysis of the sample. High-performance size-exclusion chromatography has been used in conjunction with ultraviolet detection for the molecular species and graphite furnace atomic absorption spectrometry for copper. A TSK G3000 SW column was used with an eluent consisting of Na2HPO4-NaH2PO4. The analysis of soil solutions extracted by centrifugation using this system enabled the separation of the soluble organic matter into fractions of high and low relative molecular mass. The determination of copper in these fractions revealed that 60–80% of the copper is bound to humic and fulvic acids and is unavailable for plant uptake. The proportion of copper complexed with organic matter of high relative molecular mass was found to be affected by the drainage status of the soil.

Identification of an Alveolar Macrophage-derived Activity in Bronchial Asthma that Enhances Leukotriene C4Generation by Human Eosinophils Stimulated by lonophore A23187 as a Granulocyte-macrophage Colony-stimulating Factor

Incubation of eosinophils (EOS) with alveolar macrophage (AM) supernatants isolated from asthmatic subjects followed by stimulation with the calcium ionophore A23187 resulted in enhancement of the capacity of EOS to elaborate leukotriene C4 (LTC4) (mean enhancement 169 +/- 37%, n = 31). Pretreatment of EOS with AM supernatants derived from normal individuals did not enhance LTC4 generation as compared with control medium. Enhancement was maximal when EOS were preincubated with a 1:6 dilution of AM supernatants for 5 min at 37 degrees C and were then stimulated with 5 microM A23187 for 15 min. Separation of AM supernatants by size-exclusion HPLC using a TSK G3000 SW column resulted in a peak of enhancing activity with an estimated molecular mass of approximately 30,000 D. Further purification by anion exchange HPLC using a TSK DEAE 5PW column (pH 7.4) resolved the activity into a minor peak at 0.17 M NaCl and a major peak at 0.2 M NaCl. The activities were distinct from interleukin-1 and tumor necrosis factor. Resolution of the major peak of activity by reverse-phase HPLC using a C18 spherisorb ODS column and a slope gradient of 0 to 100% acetonitrile/0.1% trifluoroacetic acid demonstrated a single peak of activity that eluted at 41% acetonitrile. The enhancing activity was sensitive to trypsin and heat and was neutralized by a specific antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF). Pretreatment of EOS with recombinant GM-CSF primed the cells for enhanced LTC4 generation following subsequent stimulation with A23187. GM-CSF may play a role in the amplification of the eosinophilic inflammation in asthmatic airways.

Specificity of milk-clotting enzymes towards bovine κ-casein

Limited proteolysis of bovine kappa-casein has been investigated with porcine pepsin A and C, and with the 2 microbial proteinases Mucor miehei proteinase and Endothia parasitica proteinase. The liberated C-terminal glycomacropeptide of kappa-casein was isolated after precipitation in 3% trichloroacetic acid followed by high-performance gel-permeation chromatography on a TSK G3000 SW column. From amino acid analyses and N-terminal sequencing of the liberated peptide it is concluded that porcine pepsin A, C and Mucor miehei proteinase cleave the same bond as chymosin: Phe-105-Met-106 whereas Endothia parasitica proteinase cleaves the bond Ser-104-Phe-105.

Heterogeneity of big-big hPRL in hyperprolactinemia.

Sera from a patient with macroprolactinoma (case 1) and from a hyperprolactinemic woman with regular menstruation (case 2) were analyzed for prolactin activity by gel filtration using Sephadex G-100, Sephadex G-200 and TSK G3000SW columns. The chromatographic profile by Sephadex G-100 showed that the percentage of immunoreactive big-big hPRL was 10.7% in case 1 and 64.1% in case 2. On Sephadex G-200 and TSK G3000SW columns, the molecular weight of big-big hPRL was estimated to be more than 500,000 daltons (big-big1 hPRL) in case 1 and approximately 250,000-300,000 daltons (big-big2 hPRL) in case 2. Big-big1 hPRL in case 1 was converted to big and little hPRLs when the serum was treated with 2-mercaptoethanol (2-ME), but part of the big-big2 hPRL in case 2 was converted to a larger molecule. Radioactive big-big hPRL generated by mixing labeled hPRL with the serum from case 1 was eluted with the void volume on Sephadex G-100 column and was not converted to the other molecular forms after 2-ME treatment. There were two radioactive big-big hPRL on TSK G3000SW column and these estimated molecular weights were more than 300,000 daltons. The data demonstrated the existence of at least two forms of big-big hPRL in the serum and indicated that radioactive big-big hPRL may be different from these hPRLs in the serum.

Preparative purification of functional bacteriorhodopsin by high-performance size-exclusion chromatography

High-performance size-exclusion chromatography (HPSEC) was used to produce stable bacteriorhodopsin essentially free of native lipids. The purified bacteriorhodopsin was shown to be hgihly functional when reconstituted into phospholipid vesicles. Purple membrane was washed in the detergent 3-[(3-cholamidopropyl)dimethylammonio]-2,2-hydroxy-1-propanesulfonate (CHAPSO) to remove a large fraction (65%) of the membrane lipids, solubilized in Triton X-100 and purified on a Bio-Sil TSK G3000SW column using a CHAPSO mobile phase. Pooled column fractions of bacteriorhodopsin from 25-mg sample loads show a 280 548 nm absorbance ratio of 1.5-1.6 and contain less than 4% endogenous lipids. This HPSEC method requires much less expensive synthetic detergent and is much faster than open column methods [ cf. L.J.W. Miercke, P.E. Ross, R.M. Stroud and E.A. Dratz, J. Biol. Chem., 264 (1989) 7531–7535].

Purification of tryptophan decarboxylase from a Catharanthus roseus cell suspension culture

Tryptophan decarboxylase (TDC) (E.C. 4.1.1.28) was purified from a cell suspension culture of Catharanthus roseus. All steps were carried out in buffers of pH 7.0–7.5, as the enzyme showed the highest stability within this pH range. After ammonium sulphate precipitation (40–55% saturation) from a crude cell-free extract, the enzyme was purified by hydrophobic interaction chromatography on Phenyl-Sepharose CL-4B, anion-exchange chromatography on Q-Sepharose Fast Flow and high-performance size-exclusion chromatography (SEC) on a GF-450 column coupled to a TSK G3000SW column. The enzyme appeared to be unstable, particularly in acidic medium. As determined by SEC, native TDC had an M r of about 85 000. Native gel electrophoresis showed the appearance of one major band corresponding to M r 96 000. Under denaturing gel electrophoresis conditions, the TDC band corresponded to an M r of about 49 000. Antiserum against the M r 49 000 protein also reacted strongly with two other proteins of M r 33 000 and 17 000. It is concluded that the enzyme consists of two subunits of M r 49 000 and that the two smaller proteins are degradation products of TDC.

A direct high-performance liquid chromatography assay of the enzymatic activity of enterokinase (enteropeptidase)

Bovine enterokinase (enteropeptidase) activates trypsinogen to trypsin at pH 8.0. In the presence of chicken ovomucoid, a stable complex of ovomucoid-trypsin is produced, inactivating trypsin and eliminating autoactivation of trypsinogen. The molecular size of trypsin (24,000 Da) is increased twofold on forming the ovomucoid-trypsin complex (52,000 Da). Size-exclusion chromatography on a Toya Soda TSK G2000SW column in an HPLC system and with computer-assisted analyses gives a direct quantitative determination of the amount of substrate (trypsinogen) and product (ovomucoid-trypsin). The rate of disappearance of substrate is equal to the rate of formation of product in agreement with kinetic theory. The simultaneous determination of both rates increases the reliability of the assay. The HPLC assay has an extended linear range for the velocity of the activation process as a function of enzyme concentration. The assay is reliable and accurate for highly purified preparations, samples at different steps in the purification scheme, and for a direct assay of the intestinal contents. The assay should be useful in clinical analyses.

Purification, characterization, and primary structure of Escherichia coli protease VII with specificity for paired basic residues: identity of protease VII and OmpT

Escherichia coli cells were found to contain a novel outer membrane-associated protease, designated protease VII (K. Sugimura and N. Higashi, J. Bacteriol. 170:3650-3654, 1988). This enzyme was purified to homogeneity and exhibited an apparent molecular weight of 36,000 on sodium dodecyl sulfate gels and 180,000 on a TSK G-3000SW column in the presence of Triton X-100. It was capable of cleaving several peptides at the center of paired basic residues but not at single basic residues, implying that it is distinct from trypsinlike proteases. Protease VII was most active at pH 6.0 and was sensitive to a serine protease inhibitor, diisopropylfluorophosphate, and to the bivalent cations Zn2+, Cu2+, and Fe2+. The nucleotide sequence of a protease VII gene-carrying DNA fragment, which had been cloned by complementation analysis (K. Sugimura, Biochem. Biophys. Res. Commun. 153:753-759, 1988) was determined. It carried two putative promoter regions and a putative Shine-Dalgarno sequence in addition to the complete structural gene, which encoded pre-protease VII of 317 amino acid residues, with the N-terminal 20 residues being a signal peptide. By comparing their amino acid sequences, protease VII and OmpT, which specifically cleaves ferric enterobactin receptor protein, were found to be identical.

Isolation and analysis of peptidic fragments of α-gliadin using reversed-phase high-performance liquid chromatography

Peptidic fragments of alpha-gliadin were obtained by peptic-tryptic-pancreatic (PTP) digestion of the alpha-gliadin fraction isolated by ion-exchange chromatography on a sulphopropyl-Sephadex C-50 column. The proteolytic digest was fractionated by ultrafiltration into three subfractions, PTPa1-PTPa3. The subfraction PTPa2 was then analysed and individual peaks were separated using reversed-phase high-performance liquid chromatography (RP-HPLC) using a gradient of acetonitrile in 0.1% trifluoroacetic acid and a Separon SGX-C18 sorbent. A 100-mg amount of the PTPa2 subfraction was separated in a single analysis by preparative RP-HPLC and twenty peaks were obtained for further characterization. The molecular mass in range 300-3000 was established for individual peptidic fragments by gel-permeation chromatography on a TSK-G2000 SW column.

Purification and characterization of lymphocytic clonal growth factor (LCGF) derived from human-human hybridoma SH-76 cells

Human-human hybridoma SH-76 cells were found to produce a factor that supported the growth of lymphocytic cells at low densities. The factor was purified from serum-free conditioned medium of the hybridoma cells by a successive application of ammonium sulfate precipitation, DEAE-Toyopearl, TSK G3000 SW and DEAE-5PW column chromatograph. The purified factor was a 72K single protein. The factor showed marked growth stimulating effect on lymphocytic cell lines, but had no effect on the growth of human adhesive cancer cell lines. Thus, the factor is a lymphocytic clonal growth factor (LCGF), as found previously in human plasma (Miyata, 1988). The LCGF of SH-76 cells could be produced in growth factor-free RPMI medium and purified easily from the conditioned medium. The factor is inactivated by heating at over 80°C, but is much more stable than the LCGF in human plasma.

Method for isolation of MMP-7-ase isoenzymes from rat muscle tissue extract.

Two MMP-7-ase isoenzymes were purified 100-fold from rat muscle extract to apparent homogeneity, with an overall yield of 10%, using homogenization, ultracentrifugation, high-performance aqueous size-exclusion and high-performance anion exchange chromatography methods. When using a TSK G-2000SW column, the separation resulted in a 6-fold purification and 30% recovery of isoenzymes B and C. This concentrated enzyme extract was then passed through a TSK-DEAE-2SW column, using salt gradient at pH 7.5, with an additional 25-fold purification and 90% recovery of the isoenzymes. Two symmetrical enzyme peaks, representing isoenzymes B and C, were detected when performing purity tests of the active enzymes on the anion exchanger and reversed-phase HPLC columns. The procedures involved are extraction, ultracentrifugation, chromatographies and enzyme assays and require less than five hours.

Malondialdehyde-containing proteins and their relationship to vitamin E.

A high molecular weight (Sephadex G-15 void volume), water-soluble, fluorescent material that was found to increase significantly in the mouse liver in response to vitamin E deficiency was separated into six proteins by high performance liquid chromatography (HPLC) using a TSK G2000 SW column. One of these proteins increased significantly in concentration due to vitamin E deficiency and had a molecular weight of 20,000 daltons. This protein was found to contain malondialdehyde, an end product of lipid peroxidation, attached to it presumably in a Schiff-base type structure with amino groups. This appears to be the first report in the literature of direct evidence that malondialdehyde is attached to protein in vivo.

A direct microassay for serum retinol (vitamin A alcohol) by using size-exclusion high-pressure liquid chromatography with fluorescence detection

Serum retinol (bound to plasma retinol-binding protein, RBP) can be determined by direct injection of as little as 20 μl of serum or plasma by using size-exclusion high-pressure liquid chromatography (SE-HPLC) with fluorescence detection. Toyo Soda TSK G-3000SW columns (0.75 × 7.5-cm guard column plus 0.75 × 30-cm analytical column) were eluted with 0.2 m NaCl/0.01 m phosphate buffer (pH 6.8) at 1 ml/min, with detection at 280 nm for protein elution. Fluorescence of the retinol-RBP complex was monitored with excitation at 334 nm (interference filter) and emission at 425 nm (long-pass filter). The retinol-RBP complex eluted as two peaks, the holo-RBP-transthyretin complex (apparent molecular weight 70,000) and holo-RBP (apparent molecular weight 9000). Identities of these peaks were established by immunodiffusion assay of the proteins and by extraction and analysis of retinol. Nonideal interactions with the column packing seem to be responsible for the low apparent molecular weight of holo-RBP. The first peak predominated when large volumes of serum (100 to 250 μl) were injected, and the second when small volumes (5 to 50 μl) were analyzed. The integrated area of the two fluorescence peaks due to retinol bound to RBP was proportional to the volume of a serum sample injected over the range 5 to 250 μl. Rat serum samples were analyzed for retinol by SE-HPLC and by conventional extraction and HPLC analysis; over the range 4 to 67 μg retinol/dl serum (0.14 to 2.34 μ m), a linear relationship was observed between the results of the two assay techniques ( n = 25, r = 0.93). Reproducibility was ±4% within-day, ±6% between days. At a signal:noise ratio of 5:1, the minimum amount of retinol quantitable was 1 ng, corresponding to 5 μg retinol/dl (0.17 μ m) for a 20-μl injection.

高速液体クロマトグラフィー法によるアポ蛋白の単離と分析

Using the molecular sieve high performance liquid chromatography (HPLC), we analyzed human serum apolipoproteins and attemped to isolate them. The apolipoproteins obtained from human serum very low-density lipoproteins (VLDL), high-density lipoproteins (HDL) and lipoprotein depleted serum were chromatographed on TSK G3000SW column. HPLC was performed in 6M urea (pH3.15) or 0.1M Tris-buffered 4M guanidine hydrochloride (pH7.0). As a result, better separation of apoproteins were achieved using 4M guanidine hydrochloride as an elution buffer. By this technique, apo VLDL could be separated into three peaks of apo B, apo E and apo C, apo HDL into apo E, apo A-I, apo A-II and apo C, and the apoproteins of d>1.21g/ml fraction into albumin, apo A-IV and apo A-I. Pure materials of apo E, apo A-I and apo A-IV could be obtained by a single column operation.

High-performance liquid chromatography-inductively coupled plasma profiles of cadmium, zinc, sulphur and other elements in rat liver supernatants after cadmium injection

The distribution of cadmium zinc sulphur and other elements in the liver supernatants of rats and their variation with time following a single intraperitoneal injection of cadmium chloride were characterized by high-performance liquid chromatography (HLPC) coupled with an inductively coupled plasma. Cadmium that was bound to high-molecular-weight-proteins before the induction of metallothionein was analysed more easily using an Asahipak GS-520 column whereas the distributions of other elements and of cadmium bound to metallothionein were better characterized on a TSK G3000SW column. Cadmium bound primarily to a distinct high molecular-weight protein before the induction of metallothionein. After the injection its distribution shifted with time to metallothionein. An atomic emission spectrometer employing an inductively coupled argon plasma and a vacuum UV monochromator was effectively used as a multi-element specific detector for HPLC.

Isolation and sequencing of α-tubulin peptides from myxamoebae of the slime mould Physarum polycephalum

Starting from only 5.9 mg of α-tubulin from myxamoebae of the slime mould Physarum polycephalum, we have isolated and sequenced peptides that account for 96% of the complete sequence. The peptides were generated by digestion of α-tubulin with trypsin, Staphylococcus aureus protease and cyanogen bromide. They were then separated according to size on a TSK G2000 SW column using a 10 mM ammonium acetate buffer at pH 6.8. In addition to good peptide separations, a time-consuming desalting step with subsequent loss of material was unnecessary because the relatively small amount of ammonium acetate could be removed by lyophilisation. High resolution of peptides from the TSK fractions was achieved on C4 or C18 reverse-phase columns by eluting with a gradient of acetonitrile in 50 mM ammonium acetate (pH 6.8) and in 0.1% trifluoroacetic acid, respectively. The peptides were then sequenced using a gas phase sequencer.

Enzymatic synthesis of lignin: Purification to homogeneity of the three O-methyltransferases of tobacco and production of specific antibodies

Three o-diphenol- O-methyltransferases (OMTs; EC 2.1.1.6) involved in the biosynthesis of lignin have been purified to homogeneity from tobacco leaves. Seven different fractionation steps which included (NH 4) 2 SO 4 precipitation, conventional low-pressure chromatography on Ultrogel AcA34 and DEAE-cellulose columns, high-performance liquid chromatography (HPLC) on three different supports (Mono Q, Mono P, and TSK G-3000 SW columns), and finally preparative electrophoresis were necessary. At each step of purification, the protein content of the enzymatic fractions was analyzed by electrophoresis on polyacrylamide gels under denaturing conditions. Purified OMT I appeared on sodium dodecyl sulfate-polyacrylamide gel as a doublet with electrophoretic mobilities corresponding to molecular weights of 38,500 ± 2000 and 39,500 ± 2000. The other two enzymes migrated as single but rather broad bands with molecular weights of 42,000 (OMT II) and 43,000 (OMT III). Polyclonal antibodies were raised in rabbits. The titers of antibodies were measured by an indirect enzyme-linked immunosorbent assay method, and their specificity was demonstrated by immunoblotting enzyme preparations at different stages of purification. Immunodetection of the three enzymes with a specific antiserum suggested serological relationships between the three OMTs of tobacco.

Purification and characterization of a novel metalloendopeptidase from Streptococcus cremoris H61. A metalloendopeptidase that recognizes the size of its substrate.

An endopeptidase (LEP-II), which has a unique substrate specificity, was purified to homogeneity by conventional chromatographic techniques from Streptococcus cremoris H61. The enzyme was a metalloendopeptidase since it was inhibited by EDTA and 1,10-phenanthroline; the metal-depleted enzyme could be fully reactivated by micromolar levels of Zn2+ and was not inhibited by specific inhibitors for serine or thiol protease. The molecular mass of the enzyme was estimated to be 80 kDa by Sephacryl S-300 gel filtration and high-performance liquid chromatography with a TSK-G3000SW column. The enzyme consisted of two identical subunits and the N-terminal sequence of LEP-II was determined up to the 19th residue. Although the enzyme had a broad substrate specificity it specifically hydrolyzed the peptide bonds involving the amino groups of hydrophobic amino acid residues. Various small polypeptides, such as alpha s1-CN(f1-23), alpha s1-CN(f91-100), oxidized insulin B chain, glucagon and some biologically active peptides were hydrolyzed. However, a variety of larger polypeptides or proteins, such as alpha s1-CN(f1-54), alpha s1-CN(f61-123), alpha s1-CN(f136-196), alpha s1-casein, beta-casein, and kappa-casein were not hydrolyzed. LEP-II recognized the size of its substrates, which were limited below a molecular mass of about 3.5 kDa.

High-performance size-exclusion chromatography of polypeptides on a TSK G2000SW column in acidic mobile phases

Polypeptides subjected to high-performance liquid chromatography on a TSK G2000SW column in acidic mobile phases of low ionic strength underwent a combination of size exclusion and ion exclusion from the matrix. The distribution coefficient ( K d) of each polypeptide was dependent upon the concentration of ion-pairing agent present and increased as the concentration of ion-pairing agent increased. The fractionation range of the column could thus be manipulated by altering the concentration of acid in the mobile phase. The nature of the ion-pairing acid, in terms of hydrophilic or hydrophobic ion pairing, also had an effect on the chromatography, such that hydrophobic ion-pairing agents lead to increased K d values, especially in the case of the most basic polypeptides. To account for these results it is proposed that the TSK GSW matrix is cationic at low pH, and that ion pairing suppresses the ion exclusion experienced by cationic polypeptides under these conditions.

Purification and characterisation of the fourth component of bovine complement.

A relatively rapid method for the isolation of complement protein C4 from bovine plasma is described. The method consists of DEAE Sephacel anion exchange chromatography of plasminogen-depleted bovine plasma followed by cation exchange chromatography on CM Sepharose and finally gel filtration on a TSK G3000 SW column. A yield of approximately 20% was obtained. Conventional SDS-PAGE of purified bovine C4 showed the presence of alpha, beta and gamma polypeptide chains, the molecular weights of which were determined from Ferguson plots to be 95,000 +/- 2,500, 80,500 +/- 2,000 and 30,000 +/- 500 daltons, respectively. SDS-PAGE of C4 immunoprecipitated from the plasma of individual cattle in gels with a reduced proportion of crosslinker showed size polymorphism of the alpha chain. The presence of dual alpha chains was confirmed by radiolabelling their reactive thiol ester moiety with 14C methylamine. The difference in size of the two bovine alpha chains is approximately 1,800 daltons. On activation of bovine C4 both alpha chains were cleaved into alpha' chains (87,000 and 85,000 daltons) characteristic of C4b.