A method for estimating the rejection coefficient-molecular weight relationship of ultrafiltration membrane for a chain polymer by using gel permeation chromatography

An improved method is presented for estimating rejection coefficient-molecular weight relationship of an ultrafiltration membrane for a polydisperse chain polymer. It is based on the basic idea using gel permeation chromatography originally developed by Cooper and Van Derveer. The method, in which peak spreading of an elution curve of the polymer was taken into consideration, is available for evaluating the relationship over a wide range of the molecular weight through only one experiment in analyses of the retentate and filtrate.     

Separation of enzymatic activities in Dictyostelium discoideum by high-pressure gel permeation chromatography.

High-pressure gel permeation chromatography was used to separate the cyclic AMP phosphodiesterase and ATP pyrophosphohydrolase activities of Dictyostelium discoideum. Two types of column packings, with different functional groups on the silica-bonded carbon side chains, were used to separate the two activities in approximately the same amount of time and with the same elution pattern. Recovery of both activities was enhanced when acetate, rather than sulfate, was the mobile phase. This recovery of activity following chromatography at high pressure demonstrates that high-pressure gel permeation chromatography can be used for the purification of enzymes.     

High-performance aqueous gel permeation chromatography of serum lipoproteins: Selective detection of cholesterol by enzymatic reaction

A rapid method for the quantitation of cholesterol in each lipoprotein fraction has been developed which utilizes high-performance aqueous gel permeation chromatography followed by enzymatic reaction using reaction-type high-performance chromatography.Cholesterol in serum lipoproteins eluted from the column could be sensitively and selectively detected by the absorbance at 550 nm following the enzymatic reaction. The sensitivity of the detection for cholesterol measured by A₅₅₀ was compared with that for protein measured by A₂₅₀ using the standard lipoprotein fractions: low-density lipoprotein (LDL) and high-density lipoproteins (HDL₂ and HDL₃). The effects of changing the flow-rate and lengthening the column on the resolution of LDL and HDL were examined. Analyses of serum protein and cholesterol were performed with this method for human and animal subjects.     

Analysis of mycotoxins in cereals and animal feed using a multi-toxin gel permeation chromatography clean-up method

Gel permeation chromatography has been used to clean up extracts from cereals and animal feeds containing a range of mycotoxins. A mixture of dichloromethane: IM hydrochloric acid, 10:1 by volume is used as the extraction solvent and clean-up is carried out on a Bio — Beads S-X3 column using dichloromethane: ethyl acetate containing a small amount of formic acid as the elution solvent. Chromatographic separation and detection was by HPLC with fluorescence or ultraviolet detection although the choice of detection method is left to the user. The method has been tested for 14 mycotoxins and results are presented for cereals fortified with mycotoxins and for samples naturally contaminated with aflatoxins, citrinin, zearalenone, and ochratoxin A.     

Kinetic investigation of the inhibitory effect of gemcitabine on DNA polymerization catalyzed by human mitochondrial DNA polymerase

Gemcitabine, 2'-deoxy-2', 2'-difluorocytidine (dFdC), is a drug approved for use against various solid tumors. Clinically, this moderately toxic nucleoside analog causes peripheral neuropathy, hematological dysfunction, and pulmonary toxicity in cancer patients. Although these side effects closely mimic symptoms of mitochondrial dysfunction, there is no direct evidence to show gemcitabine interferes with mitochondrial DNA replication catalyzed by human DNA polymerase gamma. Here we employed presteady state kinetic methods to directly investigate the incorporation of the 5'-triphosphorylated form of gemcitabine (dFdCTP), the excision of the incorporated monophosphorylated form (dFdCMP), and the bypass of template base dFdC catalyzed by human DNA polymerase gamma. Opposite template base dG, dFdCTP was incorporated with a 432-fold lower efficiency than dCTP. Although dFdC is not a chain terminator, the incorporated dFdCMP decreased the incorporation efficiency of the next 2 correct nucleotides by 214- and 7-fold, respectively. Moreover, the primer 3'-dFdCMP was excised with a 50-fold slower rate than the matched 3'-dCMP. When dFdC was encountered as a template base, DNA polymerase gamma paused at the lesion and one downstream position but eventually elongated the primer to full-length product. These pauses were because of a 1,000-fold decrease in nucleotide incorporation efficiency. Interestingly, the polymerase fidelity at these pause sites decreased by 2 orders of magnitude. Thus, our pre-steady state kinetic studies provide direct evidence demonstrating the inhibitory effect of gemcitabine on the activity of human mitochondrial DNA polymerase.     

Purification of component A of the soluble methane monooxygenase of Methylococcus capsulatus (Bath) by high-pressure gel permeation chromatography

A major improvement in the purification of the oxygenase protein (component A) of the methane monooxygenase has been effected. By employing high-pressure gel permeation chromatography several purification steps may be omitted from the previously published scheme. Furthermore the yield of the protein is enhanced and more importantly the recovered protein displays an increased specific activity, unlike that purified by other techniques.     

Molecular weight of tumor necrosis factor determined by gel permeation chromatography alone or in combination with low-angle laser light scattering

The molecular weight of human recombinant tumor necrosis factor was determined at neutral pH by gel permeation chromatography alone or in combination with low-angle laser light scattering. Mean values of 39,200 +/- 800 and 48,800 +/- 900, respectively, were obtained by the two analyses. The results resolve the apparent discrepancy in the reported values of the molecular weight of this cytokine, confirming that it exists as a trimer in neutral solution with a molecular weight of about 50,000.     

Partial amino terminal sequence of the precursor of mitochondrial ATPase inhibitor protein synthesized with mRNA partially purified by gel permeation chromatography

Messenger RNA coding mitochondrial ATPase inhibitor protein, a small peptide comprised of 63 amino acid residues, was separated from a large quantity of mRNAs of larger molecules by high speed gel permeation chromatography. Messenger RNA coding a small stabilizing factor of inactivated F1F0-ATPase complex, which is also comprised of 63 amino acids, was recovered in the same fraction as the ATPase inhibitor, whereas mRNA for a large stabilizing factor with an apparent molecular weight of 15,000 was recovered in a fraction of slightly larger molecules. ATPase inhibitor precursor labeled with various kinds of radioactive amino acids was prepared separately by cell-free translation with the purified mRNA, and the amino terminal sequence of the precursor was examined. It was demonstrated that an extra peptide of 21 amino acid residues, including 5 leucine, 4 serine, 1 glycine, and 1 methionine residues, is located at the amino terminus of the ATPase inhibitor precursor.     

Heat-induced aggregation of recombinant erythropoietin in the intact and deglycosylated states as monitored by gel permeation chromatography combined with a low-angle laser light scattering technique.

Aggregate formation of recombinant human erythropoietin (r-EPO) on heat-treatment was followed by gel permeation chromatography combined with a low-angle laser light scattering technique under various conditions with respect to pH and salt concentration in order to provide basic knowledge about the change strictly required to be monitored for medicinal proteins. When heated at 60 degrees C at neutral pH, an aggregate with a limited size consisting of about 20 r-EPO molecules was formed. On heating at 50 degrees C at acidic pH, aggregation was unlimited. The aggregation proceeded non-covalently in acidic pH, but in alkaline pH covalent bond formation was also involved. Increase in salt concentration enhanced the aggregation. Deglycosylation of the N-linked oligosaccharides made r-EPO remarkably susceptible to aggregation on heat-treatment, indicating that the carbohydrate chains are essential to the stability of r-EPO.     

Dynamic structure of vesicle-bound melittin in a variety of lipid chain lengths by solid-state NMR

Solid-state 31P- and 13C-NMR spectra were recorded in melittin-lecithin vesicles composed of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Highly ordered magnetic alignments were achieved with the membrane surface parallel to the magnetic field above the gel-to-liquid crystalline phase transition temperature (Tc). Using these magnetically oriented vesicle systems, dynamic structures of melittin bound to the vesicles were investigated by analyzing the 13C anisotropic and isotropic chemical shifts of selectively 13C-labeled carbonyl carbons of melittin under the static and magic-angle spinning conditions. These results indicate that melittin molecules adopt an alpha-helical structure and laterally diffuse to rotate rapidly around the membrane normal with tilt angles of the N-terminal helix being -33 degrees and -36 degrees and those of the C-terminal helix being 21 degrees and 25 degrees for DLPC and DPPC vesicles, respectively. The rotational-echo double-resonance method was used to measure the interatomic distance between [1-13C]Val8 and [15N]Leu13 to further identify the bending alpha-helical structure of melittin to possess the interhelical angles of 126 degrees and 119 degrees in DLPC and DPPC membranes, respectively. These analyses further lead to the conclusion that the alpha-helices of melittin molecules penetrate the hydrophobic cores of the bilayers incompletely as a pseudo-trans-membrane structure and induce fusion and disruption of vesicles.     

Treatment of olive mill waste-water by aerobic biodegradation: an analytical study using gel permeation chromatography, ultraviolet-visible and Fourier transform infrared spectroscopy

Liquid waste from olive oil mills was digested following inoculation with soil microorganisms and fractionated through various grades of gel. The fractionation showed the range of sizes of the molecules in the waste. In addition, the disappearance of the low molecular weight fraction, which is retained by the gel, and the increase of the high molecular weight fraction, which is excluded by the gel, during the last stages of the microbial treatment, indicates polymerisation of the low-molecular-weight subunits. Characterization of the fractions by UV-visible and FTIR spectroscopy confirmed the increase in their degree of polymerisation during the treatment. This is paralleled by a reduction in the amount of aliphatic components and a concomitant increase in aromatic structures.     

Lactoferrin-melanin interaction and its possible implications in melanin polymerization: crystal structure of the complex formed between mare lactoferrin and melanin monomers at 2.7-A resolution

The concentration of melanin determines the intensity of colors of the skin and hair of animals. Melanin pigments are tyrosine-based polymers formed in melanocytes within specialized organelles called melanosomes. In order to understand the mechanism of melanin polymerization, lactoferrin, a basic protein with a pI value of 9.0, has been used to produce melanin. Lactoferrin is a monomeric iron-binding protein with a molecular weight of 80 kDa. The crystals of lactoferrin were soaked in a solution containing dihydroxyphenylalanine (DOPA) and tyrosinase enzyme. These crystals were used for X-ray intensity data collection. The intensity data were collected to 2.7-A resolution to an overall completeness of 91% with an R(sym) of 0.071. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with cell dimensions: a = 85.0 A, b = 99.8 A, c = 103.4 A. The structure was determined by molecular replacement method, using the model of diferric mare lactoferrin, and refined to an R-factor 0.215 (R(free) = 0.287) for all the data to 2.7-A resolution. The final model comprises 5,281 protein atoms from 689 amino acids, 2Fe(3+), 2CO(2-)(3) ions, 2 indole-5,6-quinone molecules (IQ), and 73 water molecules. Two IQ molecules, one in each lobe, bind to lactoferrin. In the C-lobe, the IQ binds in the iron-binding cleft, whereas in the N-lobe, it is located in the side pocket between two alpha-helices, filled with solvent molecules in the native iron-saturated mare lactoferrin. The IQ molecules interact with protein molecule mainly through glutamic acid in both lobes, without significant perturbation to the protein structure. The orientation of N- and C-lobes in the present structure is similar to that observed in the native iron-saturated protein. However, as a result of the binding of IQ molecules, the orientations of the domains N1, N2 and C1, C2 in the two cases differ slightly.     

Behavior of the inadvertent pH transient formed by a salt gradient in the ion-exchange chromatography of proteins

The inadvertent pH transient produced when a stepwise change in salt concentration is used as the eluent in ion-exchange chromatography was studied theoretically using a local-equilibrium theory and experimentally using both strong-base and weak-base anion-exchange column packings. The accuracy of the local-equilibrium theory was verified by comparing it to a full numerical solution of the governing partial differential equations obtained using the method of characteristics. The predictions from the local-equilibrium theory were observed to largely agree with experimental results. Detailed comparisons of experimental results and the local-equilibrium theory permitted the observed trends for the pH transients to be interpreted in terms of the physical properties of the column packing and mobile phase. The results of this study are useful for the design of ion-exchange processes using salt gradient elution where it is desired to limit the exposure of eluted proteins to the inadvertent pH transient caused by the salt gradient.     

Human fibrinogen gel formed by the action of a cell surface polysaccharide obtained from a Staphylococcus

An alkali-stable polysaccharide (called compact-colony forming active substance; substance 1) obtained from the cell surface of a strain of Staphylococcus epidermidis caused gel formation of human fibronogen, with no release of fibrinopeptides. Substance 1 possessed neither esterase nor caseinolytic activities; no inhibition of gel formation was shown by dinitrofluorophosphate. Heparin and galactose prevented gel formation of fibrinogen with substance 1. With the addition of early- and late-fibrinogen or fibrin degradation products into the fibrinogen sample, no prolongation of the gel formation time was observed. This substance is, therefore, assumed to nonenzymatically induce gel formation with fibrinogen, a process resembling paracoagulation.     

Polar head molecular packing of dipalmitoylglycerophosphocholine in the gel state: a fluorescence investigation

Binding of the fluorescent probe 8-anilino-1-naphthalenesulfonate to lecithin monolayers was shown to be dependent on the molecular packing of the lipids. No binding was observed in the gel state. The binding site appeared to be structurally unaffected during the compression of the monolayer, and it was concluded that the site was an assembly of "fluid" lipids. Four lipids are in close contact with the probe, and they are surrounded by 8-10 "fluidized" others. From these observations, the dissociation constant appeared as a good indicator of molecular packing at the polar head level in other lipid assemblies. In the gel state, macrovesicles and multilayers are a tight homogeneous assembly; at the same temperature, microvesicles display defects, in a highly fluid state, which are embedded in a rigid matrix where no binding occurs. Ten percent of the outer layer are in this "nongel" organization.     

Synthesis of a polymer skeleton at the inner leaflet of liposomal membranes: polymerization of membrane-adsorbed pH-sensitive monomers

We describe the synthesis of liposomes with an artificial membrane skeleton as a model of the native cellular cytoskeleton. Similar to natural conditions, a flat polymer network is coupled to the inner membrane leaflet like a suspended ceiling via membrane-inserted anchor monomers with a spacer. The polymer is composed of DMAPMA (N-(3-N,N-dimethylaminopropyl) methacrylamide) and TEGDM (tetraethylene glycol dimethacrylate) as a linker and is coupled to the membrane anchor DOGM (1,2-distearyl-3-octaethylene glycol glycerol ether methacrylate). In the first step of the synthesis, DMAPMA and TEGDM are encapsulated into liposomes composed of egg phosphatidylcholine (EPC), and free monomers are removed by gel chromatography. At pH 10, DMAPMA adsorbs to the inner membrane surface, as demonstrated in parallel studies with lipid monolayers using a Langmuir film balance. The polymerization by UV irradiation was initiated with DEAP (2,2-diethoxyacetophenone) as the initiator and was shown to be complete after 15 min. At pH 6, polymer was desorbed from the inner membrane surface to form a lamellar structure similar to that of the cellular cytoskeleton, as shown by electron microscopy. In comparison to NIPAM (N-isopropylacrylamide), which was used as a monomer in a recent study (Stauch, O.; Uhlmann, T.; Frohlich, M.; Thomann, R.; El-Badry, M.; Kim, Y.-K.; Schubert, R. Biomacromolecules 2002, 3, 324-32), DMAPMA shows much slower membrane permeation leading to an essential restriction of the formed polymer to the liposomal interior. The DMAPMA-based composite structure stabilizes the lipid membrane against sodium cholate by a factor of 2.5 as compared to plain EPC liposomes. This is discussed in the context of the situation in the liver, where the cytoskeleton probably plays a crucial role in the stabilization of the membrane against high bile salt concentration.     

GTP hydrolysis of cell division protein FtsZ: evidence that the active site is formed by the association of monomers.

The essential prokaryotic cell division protein FtsZ is a tubulin homologue that forms a ring at the division site. FtsZ forms polymers in a GTP-dependent manner. Recent biochemical evidence has shown that FtsZ forms multimeric structures in vitro and in vivo and functions as a self-activating GTPase. Structural analysis of FtsZ points to an important role for the highly conserved tubulin-like loop 7 (T7-loop) in the self-activation of GTP hydrolysis. The T7-loop was postulated to form the active site together with the nucleotide-binding site on an adjacent FtsZ monomer. To characterize the role of the T7-loop of Escherichia coli FtsZ, we have mutagenized residues M206, N207, D209, D212, and R214. All the mutant proteins, except the R214 mutant, are severely affected in polymerization and GTP hydrolysis. Charged residues D209 and D212 cannot be substituted with a glutamate residue. All mutants interact with wild-type FtsZ in vitro, indicating that the T7-loop mutations do not abolish FtsZ self-association. Strikingly, in mixtures of wild-type and mutant proteins, most mutants are capable of inhibiting wild-type GTP hydrolysis. We conclude that the T7-loop is part of the active site for GTP hydrolysis, formed by the association of two FtsZ monomers.     

Identification of a nonimmunoreactive but highly bioactive form of prolactin in the mouse pituitary by gel electrophoresis.

Polyacrylamide gel electrophoresis of fresh mouse pituitary extracts in 10% acrylamide reveals three closely situated bands of protein in the area where prolactin usually migrates. The fastest migrating band constitutes only 10–30% of the total, but it is twice as active in the pigeon crop-sac bioassay as the major band and has little or no immunologic cross-reactivity against an antiserum to the major constituent. This newly recognized band may be a hitherto unrecognized molecular variant of prolactin.