Bio-Gel Chromatography Research Articles

Middle Molecule Adsorption from Ultrafiltrate for Use in Intensive Care Medicine

Continuous renal replacement therapies, such as hemofiltration, have been used increasingly on intensive care units over the last decade as a means of managing patients with acute renal failure (1). It is clear that the regeneration of patients' ultrafiltrate (UFR), via the removal of uremic toxins, allowing it to be replaced back into the circulation, would reduce the cost burden associated with replacement solutions. This study investigated the potential use of novel, mesoporous polymer based carbons, produced by the pyrolysis and steam activation of a vinylpyridine-divinylbenzene copolymer (SCN) to remove middle molecule sized uremic toxins from UFR. Previous studies have shown that SCN has a high capacity for the adsorption of other uremic toxins including creatinine and to a lesser degree, urea (2). UFR samples were obtained from patients with acute renal failure (n=6) at an early stage in hemofiltration. The UFR samples were passed through a column containing 6g of non-oxidised SCN. Fractions were collected every 5 minutes for further analysis by Tris-Glycine SDS-PAGE gels (7.4 – 200 kD), Biogel Chromatography (>5 kD) and Tris-Tricine SDS-PAGE gels (1.5 – 26.6 kD). Both gel systems showed complete adsorption of all protein bands from the 5 minute sample with increasing break through of UFR proteins in the eluate over time. The chromatography column showed complete removal of all middle molecules from the pre-column aliquot when compared to that of the 360 minute aliquot. These results demonstrate the ability of mesoporous carbons to adsorb substances within the middle molecule rage (MW = 0.3 – 15 kD). The results presented here further support the use of activated carbons in the regeneration of UFR. The flexibility offered by these adsorbents through close control of their chemical purity, pore size distribution and surface chemistry makes them highly biocompatible and allows the targeting of specific molecules. This technology also offers potential benefits in other areas of intensive care medicine, in which specific adsorption may be of benefit, such as the removal of inflammatory molecules in sepsis.

Tc‐Glutathione Complex (Tc ‐GSH): Labelling, ChemicalCharacterization and Biodistribution in Rats

The chemical structure of 99mTc-GSH has been estabilished using the 99Tc isotope. Labeling of glutathione with technetium in the presence of stanous chloride gave a high yield result. In a comparative study between 99Tc and 99Tc glutathione, the Tc-GSH complex obtained was purified and characterized by uv, visible spectroscopy, HPLC, Biogel chromatography, mass and NMR spectroscopy. Stoichiometric analysis showed a 2 : 1 molar ratio of GSH/Tc for the reaction. The molecular mass assessed by mass spectroscopy was 727 Da corresponding to an oxo(bis) glutathione technetate. NMR studies demonstrated that each glutathione molecule was coordinated to technetium via cysteinyl sulfur and nitrogen atoms. The biodistribution of the complex was studied in normal rats. Blood clearance was rapid during the first hour involving a biexponential curve ( t1/2 (1) : 50 min, t1/2 (2) : 400 min ). No radioactive accumulation was found in any specific organ except kidney and bladder. All the activity excreted was found unchanged in urine. In conclusion, Tc-GSH displayed an anionic dimer form as GSH-Tc-GSH. We assume that the complex is a tetradentate (2N,2S) complex containing a pentavalent technetium coordinated by two thiol and nitrogen atoms of both GSH ligands, and an apical oxo group.

Chromatographic studies on the isolation of peroxydisulphate oxidation products of primaquine

Eight compounds from peroxydisulphate oxidation of primaquine were fractionated on Bio-Gel P-2 column using water as an eluent. A HPLC method employing acetonitrile–methanol–1 M perchloric acid–water (30:7:1:95, v/v) as a mobile phase at 1.0 ml/min on μBondapak reversed-phase column and UV detection at 254 nm was developed for the separation and identification of different oxidation products of primaquine. A combination of Bio-Gel chromatography with reversed-phase HPLC was found to be the most suitable analytical technique for the semipreparative isolation of various products formed from the oxidation. Two oxidation products that were isolated had three or four times higher gametocytocidal activity as compared to primaquine.

An introduction to electrospray ionization and matrix‐assisted laser desorption/ionization mass spectrometry: Essential tools in a modern biotechnology environment

The tremendous progress in biochemistry and biotechnology has been made possible in part by recent advances in analytical methods, in particular mass spectrometry. With the introduction of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), mass spectrometry allowed the determination of the molecular weight of peptides and proteins with a much greater accuracy than achievable by traditional methods such as SDS-PAGE and biogel chromatography. In addition, these mass spectrometry experiments have become routine and can be performed within minutes. ESI and MALDI (in combination with enzymatic methods) can also provide vital structural data such as the amino acid sequence and the sites of posttranslational modifications for peptides and proteins with a sensitivity that competes favorably with other methods. The use of ESI and MALDI is not limited to peptides and proteins; analysis of oligonucleotides and oligosaccharides has been simplified by these techniques as well. For information about structurally significant noncovalent interaction between various types of biomolecules, ESI is probably one of the most convenient methods. Not surprisingly, we anticipate that the mass spectrometers with these ionization capabilities will soon become standard equipment in all pharmaceutical and biotechnology laboratories. © 1997 John Wiley & Sons, Inc. Biospectroscopy 3: 259–280, 1997

Purification and Properties of NAD- and NADP-dependent Malic Enzymes from Bradyrhizobium japonicum Bacteroids

Summary Bacteroids were isolated from 6 weeks-old soybean nodules and the NAD- and NADP-dependent malic enzymes purified by ammonium sulfate precipitation, DEAE-cellulose ion exchange chromatography, Bio-Gel chromatography and fast flow Q-Sepharose ion-exchange chromatography. NADP-ME (E.C. 1.1.1.40) was purified 40-fold and NAD-ME (E.C. 1.1.1.38) 14-fold. Native PAGE demonstrated that NAD-ME and NADP-ME from bacteroids are two distinct enzymes. The molecular weight of the native enzymes was estimated by means of gel filtration and SDS-PAGE. It was shown that NADP-ME is a 60 kD monomer and NAD-ME is a heterodimer with subunits of 60 and 80 kD. NADP-ME (K M : 0.18 mM) exhibited much higher affinity for malate than NAD-ME (K M : 5 mM). The role of both enzymes in bacteroid metabolism is discussed.

Purification and characterization of an endonuclease (E.C. 3.1.30.1) from Streptomyces tendae.

A single-strand-specific endonuclease which converted negatively supercoiled DNA to open-circular and linear DNA was purified to homogeneity with Hb-Sepharose 4B, DEAE Trisacryl M, HA-Ultrogel and PBE-94 chromatofocusing from extracts of Streptomyces tendae ATCC 31160. Bio-Gel P-200 chromatography and electrophoresis in SDS-PAGE indicated the native protein was a monomer with a molecular weight of approximately 40-kDa. This enzyme did not hydrolyze double-stranded linear DNA but digested RNA and circular single-strand DNA. Sequence specificity for nicking of negatively supercoiled DNA was not detected.

Fractionation of chromatin fragments on columns of Biogel A50-m at different salt concentrations

Nuclease fragmented chromatin was chromatographed on Biogel at various NaCl concentrations. The yield of eluted chromatin, and its H1/core histone ratio was minimal at 0.18 M NaCl where the ratio of H1 subtypes H1c/H1 ab was maximal. Therefore, the eluted material was aggregation-resistant chromatin while aggregatable chromatin remained on the columns. Previous results were interpreted as Hl depletion of chromatin by ion-exchange properties of Biogel, but the primary phenomenon is now seen as a separation of classes of chromatin that differ in sensitivity to salt-induced aggregation. At very low salt concentrations, Biogel chromatography can be used without concern for H1 depletion.

Purification, preliminary characterization, and immunological comparison of hog lens leucine aminopeptidase (EC 3.4.11.1) with hog kidney and beef lens aminopeptidases

Leucine aminopeptidase (LAP) was purified from hog lenses by application of the Himmelhoch procedure for isolation of hog kidney LAP [S. R. Himmelhoch (1970), in Methods in Enzymology (Perlmann, G. E., and Lorand, L., eds.), Vol. 19, pp. 508–513, Academic Press, New York.] This involved treating crude hog lens homogenates with hexadecyltrimethylammonium bromide, DEAE-cellulose adsorption and elution, ammonium sulfate fractionation (53–84% of saturation), and gel filtration on a Bio-Gel A-1.5m column. Purifications ranging from 2080- to 4700-fold with activity yields from 28 to 100% were achieved. The hog lens LAP appeared homogeneous by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE). Bio-Gel chromatography of the native enzyme and SDS-PAGE of dimethylsuberimidate-crosslinked LAP indicated a molecular weight of 326,000. SDS-PAGE of untreated LAP showed a subunit weight of 54,000, consistent with a hexameric enzyme structure. By immunodiffusion, LAP from hog lens and kidney were identical while hog lens and beef lens enzymes demonstrated only partial identity. Electrophoresis of the native enzymes showed a slightly lower mobility for the hog lens LAP than for beef LAP at pH 8.7.

Glycosylation of a receptor specific for asialoglycoproteins in rat liver.

Glycosylation of a rat receptor specific for asialoglycoproteins was investigated in vivo by using monospecific antibody. After intravenous injection of [3H]mannose, the receptor protein was immunoprecipitated from various subcellular fractions and the glycopeptide and oligosaccharide chains of the protein were prepared by treatment with pronase and endo-beta-N-acetylglucosaminidase H. The glycopeptides thus prepared from the rough and smooth microsomes and Golgi heavy fraction (GF3) were all sensitive to endo H and most of the endo H-sensitive oligosaccharides were eluted at the position considered to correspond to Man8GlcNAc on high-resolution Bio-Gel chromatography. Endo H-resistant forms were first detected in the Golgi intermediate fraction (GF2) and significantly in the light Golgi fraction (GF1), suggesting the formation of the complex-type oligosaccharide chains in the latter fraction. This view was also supported by the almost exclusive addition of the terminal sugars such as N-acetylglucosamine, galactose, and sialic acid in GF1. These results suggest that the major form of the oligosaccharide chains from the endoplasmic reticulum to the Golgi apparatus is Man8GlcNAc2 and that the processing of these large mannosyloligosaccharide chains and subsequent addition of terminal sugars to them are performed successively in the trans-Golgi region.

Identification of a tumor inhibitory factor in rat ascites fluid

A polypeptide which inhibits the growth of human carcinoma cells has been characterized from Novikoff rat ascites fluid. This tumor inhibitory factor co-purified with transforming growth factor activity through acid/ethanol extraction and Bio-Gel chromatography. The two activities were completely separated by reverse phase HPLC. The tumor inhibitory factor is heat stable and requires disulfide bonds for bioactivity. This factor inhibited the anchorage independent growth of the more differentiated human colon carcinoma cell lines but did not affect the less differentiated carcinoma cells. The presence of stimulatory and inhibitory activities in the same extracts suggests that the relative concentrations of these factors may be important in the control of cell growth.

Intracellular forms of transferrin oligosaccharide chains in rat liver.

Glycosylation of transferrin was investigated in vivo by using antibody monospecific for rat serum transferrin. Pulse-chase experiments indicated that, after intravenous injection of [35S]methionine, labeled transferrin appeared in the rough and smooth microsomes and Golgi subfractions in rapid succession in 10 min and that an additional 10 min was required for it to be secreted. Most of the intracellular transferrin (95%) immunoprecipitated from the total microsome fraction was sensitive to endo-beta-N-acetylglucosaminidase H (endo H), whereas serum transferrin was completely resistant to it. Further fractionation of the total microsomes has revealed that the intracellular transferrin immunoprecipitated from the rough and smooth microsomes and GF3 are all endo-H-sensitive and most of the endo-H-sensitive oligosaccharides were eluted at the position corresponding to Man8GlcNAc on high-resolution Bio-Gel chromatography. This finding suggests that the major form of intracellular transferrin oligosaccharide in the course of intracellular transport from the endoplasmic reticulum to the Golgi apparatus is Man8GlcNAc2. Endo-H-resistant forms were first detected in the GF2 but more in GF1, most of which were sensitive to neuraminidase. Since the heavy Golgi subfraction contains mainly cis-Golgi elements, such as cisternae, and the light subfraction mainly trans-Golgi elements, such as secretory granules, it is strongly suggested that the processing of these large mannosyloligosaccharide chains and the subsequent addition of terminal sugars to them are performed successively in the trans-Golgi region just before secretion.

Purification of N-acetyl-L-glutamate synthetase from rat liver mitochondria and substrate and activator specificity of the enzyme.

N-Acetylglutamate synthetase was purified 33,000-fold to apparent homogeneity from the matrix fraction of rat liver mitochondria. The purification procedure involved treatment of mitochondria with digitonin, ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, Affi-Gel blue chromatography, acetylglutaminyl Bio-Gel chromatography, sucrose density gradient centrifugation, and isoelectric focusing. As the enzyme lost activity in contact with a glass surface, glassware coated with silicone was used throughout the purification. Triton X-100 stabilized the enzyme and was included in most buffers at a concentration of 0.1% (w/v). The purified acetylglutamate synthetase has a specific activity of 92.4 mumol min-1 (mg of protein)-1, in the presence of 1 mM L-arginine. The enzyme had a sedimentation coefficient (S20,w) of 8.1 S and its molecular weight was estimated to be about 160,000. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the enzyme migrated as a single protein band with Mr = 57,000, indicating a composition of three subunits of similar size. The purified acetylglutamate synthetase showed a high substrate specificity for L-glutamate and acetyl-CoA, confirming the previous results with a less purified preparation (Shigesada, K., and Tatibana, M. (1978) Eur. J. Biochem. 84, 285-291). Among other 14 acyl-CoAs tested, only propionyl-CoA could substitute for acetyl-CoA, as an acyl donor, with a reaction rate 4.3% of that with acetyl-CoA at 0.5 mM. Among amino acids other than L-glutamate and derivatives or analogues of glutamate tested, the following served as an acyl acceptor: glycine at a rate 2.7% of that with L-glutamate at 1.0 mM, L-glutamine (5.0%), L-glutamate gamma-hydroxamate (15.5%), DL-alpha-aminoadipate (5.2%), and DL-alpha-aminopimelate (4.0%). The purified enzyme is markedly stimulated by L-arginine. The specificity of L-arginine as a low molecular weight activator was strict; only L-argininic acid could activate the enzyme to a lesser extent. Cationic polypeptides, such as protamine, polyarginine and polylysine, also activated the synthetase. The effect was additive to that of arginine.

Phosphorylation on protein and carbohydrate moieties of a lysosomal arylsulfatase B variant in human lung cancer transplanted into athymic mice.

Human lung cancer transplanted into athymic mice contains predominantly an acidic variant (designated B1) of lysosomal arylsulfatase B. B1 enzyme was suggested to be phosphorylated and sialylated (Gasa, S., Makita, A., Kameya, T., Kodama, T., Koide, T., Tsumuraya, M., and Komai, T. (1981) Eur. J. Biochem. 116, 497-503). In order to determine the localization of phosphate in B1 enzyme, we labeled in vivo the transplanted tumor with [32P]H3PO4 or [3H]glucosamine and purified B1 enzyme by immunoprecipitation. Bio-Gel chromatography of the labeled B1 enzyme treated with endoglycosidase H demonstrated that both the excluded and included materials were labeled with 32P and 3H. From acid hydrolysate of the excluded materials, phosphorylated serine and threonine were detected. Protein phosphorylation of arylsulfatase was confirmed by in vitro labeling experiments with [gamma-32P]ATP. By incubation of the tumor homogenate with ATP followed by isolation of the enzymes, B1 enzyme had a significant amount of radioactivity, whereas the B enzyme had little; by exogenous protein kinase, partially purified B enzyme was phosphorylated 35 times more than B1 enzyme. Acid hydrolysate of the included materials in the Bio-Gel column demonstrated mannose 6-phosphate and an unknown phosphorylated compound which migrates more than Man-6-P on electrophoresis and chromatography.

Phosphorylation of parathyroid secretory protein.

The phosphorylation of proteins released into the medium of bovine parathyroid gland slices or isolated cells incubated with 32Pi has been investigated. The primary protein phosphorylated had a Mr of 68,000 and coeluted with newly synthesized parathyroid secretory protein (PSP) on Bio-Gel chromatography and on polyacrylamide gel electrophoresis. Isoelectric focusing of double-labeled samples ([35S]methionine and 32Pi) revealed comigration of the two radioactive markers at a pH of 4.6, which was similar to that of purified PSP. Phosphorylation of the Mr 68,000 protein was also demonstrated in cell homogenates incubated with [gamma-32P]ATP; the Mr 68,000 protein was the predominant labeled protein. Increasing quantities of calcium, with and without added EGTA, caused a progressive decrease in phosphorylation of the protein. These studies demonstrate that PSP is readily phosphorylated in parathyroid cells, that the degree of phosphorylation is inversely proportional to calcium concentration, and that PSP is the major phosphorylated protein released from the gland. The relationship of phosphorylation to the potential physiologic importance of PSP remains to be determined.

Evidence that the insulin secretagogue, β-cell-tropin, is ACTH22–39

The pituitary neurointermediate lobe of genetically obese (ob/ob) mice contains a hormone which stimulates insulin release and which cross-reacts with a -COOH-terminal ACTH antiserum, suggesting that it is related to corticotropin-like intermediate lobe peptide (CLIP), the 18-39 fragment of ACTH. The hormone, which we have called beta-cell-tropin, has been shown to be present in the plasma of the ob/ob mouse and to potentiate glucose induced insulin secretion. We have now shown that ACTH22-39 prepared by tryptic digestion of human synthetic CLIP behaves similarly on Biogel chromatography and on reverse-phase HPLC to the naturally occurring beta-cell-tropin. Furthermore, beta-cell-tropin and ACTH22-39 have indistinguishable antigenic and insulin releasing properties.

Action of β-cell tropin on insulin secretion in vivo and on lipid synthesis

A peptide of the pituitary pars-intermedia @-cell tropin) from genetically obese mice (ob/ob) stimulates insulin secretion in vitro [ 1,2]. P-Cell tropin (BCT) crossreacts with a COOH terminal ACTH antiserum. Experiments have now been carried out in which purified samples of BCT prepared by Bio-Gel chromatography [2] have been tested for in vivo stimulation of insulin release and of lipid synthesis in rats. The influence of BCT on lipid synthesis in adipocytes has also been investigated. The results show a significant rise in plasma insulin levels 2 mm following the injection of BCT into the jugular vein of rats. A significant increase in total lipid synthesis in the subcutaneous tissue of rats treated with BCT was demonstrated, 45 min following an injection of 3Hz0, as measured by ‘H incorporation into saponified fatty acids. BCT was also shown to stimulate total lipid synthesis in vitro in adipocytes incubated for 1 h.

Rat brain aryl acylamidase: Further characterization of multiple forms

1. 1. Two fractions of aryl acylamidase (EC 3.5.1.13) were further separated from rat brain extracts at pH 7.5 by ammonium sulfate precipitation and Bio-Gel chromatography. 2. 2. 1,2,3,4-Tetrahydro-β-carboline competitively inhibited (67%) fraction-1 but slightly inhibited (13%) fraction-2. Tetrahydroharman, 6-hydroxy-tetrahydroharman and harminic acid slightly inhibited both fractions. Harmalol inhibited fraction-1 but enhanced fraction-2. 6-Methoxy-harman, 6-methoxy-harmalan and harmaline enhanced both fractions. 3. 3. Pargyline did not affect either fraction. Methiothepin, cyproheptadine and chlorimipramine inhibited fraction-1 but stimulated fraction-2. 4. 4. Neostigmine moderately (30%) inhibited AAA-2 but did not have any significant effect on AAA-1. 5. 5. These results indicate that the β-carboline compounds might play a role in regulating activity of AAA-1 and 2 in brain. 6. 6. Both fractions might be related to serotonergic neurons but only AAA-2 might be associated with acetylcholinesterase.

4] Hexokinase from ascaris suum muscle

Publisher Summary This chapter describes the purification procedure and properties of hexokinase from Ascaris suum muscle. The hexokinase reaction is monitored by coupling to NADPH formation (followed by absorbance at 340 nm) with glucose-6- phosphate dehydrogenase. The muscle, which comprises 50–60% of the weight of the worm, is obtained by dissecting the worm longitudinally and removing the white reproductive tissue and the yellow-to-green intestine. The procedure includes diethylaminoethyl (DEAE)-BioGel chromatography, ammonium sulfate fractionation, and Sephadex G-200 chromatography. The purified enzyme represents the only form of hexokinase in the muscle of Ascaris saum and exhibits an apparent isoelectric point of 5.9. Both sodium dodecyl sulfate gel electrophoresis and Sephadex G-200 column chromatography give molecular weights in the range of 97,000–100,000. The inhibition constants of glucose 6-phosphate versus glucose and ATP are at least one order of magnitude higher than those for hexokinase from mammalian tissues. The product inhibition pattern of the ascarid hexokinase suggests that the enzyme has a random kinetic mechanism similar to that found in other hexokinases.

Phenoxazinone synthase from Streptomyces antibioticus: Purification of the large and small enzyme forms

Phenoxazinone synthase has been purified approximately 80-fold from 48-h-old cells of Streptomyces antibioticus. The purification procedure involves streptomycin sulfate and ammonium sulfate precipitations, affinity and Bio-Gel chromatography, and glycerol gradient centrifugation. Two forms of the enzyme are purified by these techniques, a large form (L) with a molecular weight of around 900,000 and a small form (S) with an M r of about 200,000. Electrophoresis of the large and small forms on sodium dodecyl sulfate-polyacrylamide gels reveals the presence of a single polypeptide chain with a molecular weight of around 100,000. Both L and S appear to be distinct molecular forms of the enzyme since rechromatography on Bio-Gel or recentrifugation on glycerol gradients results in retention of the mobility of the form in question. These procedures do not convert L to S or S to L. The purification procedure described has been used for the preparation of phenoxazinone synthase from 12-, 18-, and 48-h cultures of S. antibioticus. These experiments have shown that the relative amounts of L and S which can be isolated depend on the age of the cells used as starting material. As the cultures age, the relative amount of L which is present increases. This result also suggests that L and S are distinct molecular entities. Antibody to a combined L plus S preparation and to L has been raised in rabbits. Anti-L antibody reacts with both L and S and is capable of completely inhibiting phenoxazinone synthase activity in crude extracts of S. antibioticus cells. Thus, the 100,000 M r subunits of L and S must have some antigenic determinants in common, although they may not be identical in structure. Oxygen consumption during the formation of cinnabarinic acid from 3-hydroxyanthranilic acid was measured with the Clark oxygen electrode. These experiments showed that both L and S catalyze a reaction involving the consumption of 1.5 mol of oxygen per mole of phenoxazinone formed.

The Structure of Soluble Fibrin Complexes and Fibrin Degradation Products after Echis carinatus Bite

Studies on the concentration and structure of fibrinogen, fibrinogen-fibrin soluble complexes, and fibrinogen-fibrin degradation products were made on 11 patients in Nigeria who suffered defibrination following Echis carinatus bite. Following admission, before treatment with antivenom, all patients had reduced or zero fibrinogen levels, and increased concentrations of soluble complexes and degradation products. The fibrin component of the soluble complexes, separated by fibrinogen-sepharose chromatography, consisted of both intact fibrin and fibrin degraded at the alpha-chain. After isolation by Biogel chromatography the soluble complexes were also found to contain gamma-dimer chains. The fibrinogen-fibrin degradation products consisted of several X species, Y, D and D-dimer, as well as fragment E. The major fragment in all patients was D, but a few samples contained significant quantities of D-dimer, indicating in vivo activation of factor XIII. There was evidence of degraded fibrinogen, as well as fibrin, in the soluble complexes and degradation products, suggesting that fibrinogenolysis, in addition to fibrinolysis, had occurred, probably as a result of secondary endogenous activation of the fibrinolytic system in response to defibrination.