Papers by Ragnar Flengsrud
Purification and Some Characteristics of the Human Coagulation Factor VII
European journal of biochemistry, Aug 1, 1979
1. A purification procedure for factor VII (proconvertin) from human plasma is described. The pro... more 1. A purification procedure for factor VII (proconvertin) from human plasma is described. The procedure involves barium sulphate adsorption and elution. DEAE-Sephadex column chromatography, barium sulphate adsorption and elution, heparin-Sepharose column chromatography, preparative disc gel electrophoresis and finally adsorption with antiserum to prothrombin coupled to Sepharose and antiserum to albumin coupled to Sepharose. This procedure gave an approximately 8 . 10(5)-fold purification. 2. The factor VII obtained from the electrophoresis step was mainly a single-chain protein with an apparent molecular weight of 53000 +/- 2000. 3. After the final purification step, additional forms of factor VII, resulting from a fragmentation of the factor VII molecule were detected. 4. Amino acid composition data of the purified factor VII are given. 5. Antisera were raised in two different rabbits by injection of the purified factor VII. The antisera obtained gave a good titre against the factor VII activity and were not directed against any of the three other vitamin-K-dependent coagulation factors.
Procede de production d'heparine a faible masse moleculaire
La presente invention propose un procede de production d'une composition d'heparine a tre... more La presente invention propose un procede de production d'une composition d'heparine a tres faible masse moleculaire (Very Low Molecular Weight Heparin ; VLMWH) ayant une teneur en VLMWH d'au moins 10 % en masse par rapport a la teneur totale en heparine. Ledit procede comprend la reduction, par des moyens chromatographiques ou chimiques ou par filtration, de la proportion relative de l'heparine de masse moleculaire superieure a 8000 Da dans une composition d'heparine extraite d'un animal marin vascularise non mammalien.
Process for Producing Glycosaminoglycans
Characterization of production and enzyme properties of an endo-?-1,4-glucanase fromBacillus subtilis CK-2 isolated from compost soil
Antonie van Leeuwenhoek, 1994
Bacillus subtilis CK-2, isolated from garden organic waste compost, was found to have high hydrol... more Bacillus subtilis CK-2, isolated from garden organic waste compost, was found to have high hydrolytic activity against carboxymethylcellulose (CMC) due to the secretion of an endo-beta-1,4- glucanase. Enzyme production was related to the sporulation process, and was regulated by the concentration of readily metabolizable carbohydrate in growth medium. Enzyme production did not require CMC or other cellulose containing materials. The endo-beta-1,4-glucanase activity was optimal at pH 5.6-5.8 and at 65 degrees C, and achieved thermal stability up to 55 degrees C. The activity was inhibited by Hg2+. The purified enzyme gave a single band corresponding to a MW of 35.5 kDa on SDS-PAGE, while the Sephadex G-75 chromatography revealed a molecular weight of the active enzyme around 70 kDa, indicating a dimeric form of the active enzyme. The enzyme activity was irreversibly inhibited by SDS. Native PAGE and IEF revealed three different isoelectric forms of the enzyme, all with an identical N-terminal amino-acid sequence.
Biochemical journal. Cellular aspects, Aug 1, 1972
1. The supernatant obtained by centrifugation of a rat liver homogenate at 10000Og for 1 h contai... more 1. The supernatant obtained by centrifugation of a rat liver homogenate at 10000Og for 1 h contained a heat-labile macromolecular inhibitor of the thrombin-fibrinogen reaction. 2. The inhibitor was purified to electrophoretic homogeneity by repeated pre- parative polyacrylamide disc electrophoresis. Inhibition was observed with purified inhibitor equivalent to about 1 jig of protein/ml. 3. The inhibitor had a pI of 3.50-3.75, a molecular weight (from sodium dodecyl sulphate-polyacrylamide-gel electrophoresis) of 72000±3000 and was inactivated by p-hydroxymercuribenzoate or 5,5'-dithiobis- (2-nitrobenzoic acid). 4. Kinetic studies revealed a non-competitive inhibition, with the inhibitor probably acting on the thrombin-fibrinogen complex. Studies on the subcellular localization of the coagu- lation factor VII (Gaarder & Prydz, 1967, 1969) revealed the presence of an inhibitor of blood coagu- lation in the 100000g supernatant from perfused rat liver. We report here the purification of this inhibitor and some studies of its mode of action.
Biochemical journal. Cellular aspects, Dec 1, 1972
Biochemical Journal, Mar 1, 1975
Non-activated coagulation factor IX was purified approx. 10000-fold from human plasma. The final ... more Non-activated coagulation factor IX was purified approx. 10000-fold from human plasma. The final product was electrophoretically homogeneous and comprised a tingle polypeptide chain with a molecular weight ofabout 70000 and a pl of4.3-4.45. The N-terminal amino acid was glycine. The amino acid and the carbohydrate contents were analysed and a monospecific antiserum to the factor was raised in rabbits. Factor IX is a plasma protein which takes part in the intrinsic pathway of blood coagulation. Like most of the coagulation factors, factor IX exists in a zymogen form. After activation of the zymogen form by activated factor XI (XIa), the resultant activated factor IX (IXa), together with thrombin-exposed factor VIII (VlIIt), phospholipid and Ca2+ ions, causes the generation of the proteolytic enzyme, factor Xa (EC 3.4.21.6), from factor X (0sterud & Rapaport, 1970). Barton (1967) demonstrated that factor IXa, factor VIII, phospholipid and Ca2+ must be present in order to activate factor X and that the mixing of these four components did not lead to irreversible changes in any of them. It remains, however, to be clearly demonstrated that the acti- vator is a macromolecular complex, similar to the complex of factor Xa, factor V, phospholipid and Ca2+ that converts prothrombin into thrombin. To study the activation of factor IX by factor XIa, and to examine in more detail the mechanism by which factor IXa participates in the formation of factor X activator, highly purified factor IX prepara- tions with well-defined characteristics are required. We describe here a method for the purification of human factor IX. The preparations obtained were homogeneous in disc-gel electrophoresis and sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. The main difficulties in the purification were the pre- vention ofactivation offactorIX and the separation of factors IX and X. Some characteristics of the purified factor IX are reported. Chemicals and biochemicals were obtained as follows: glucose oxidase was from Boehringer Corp., Mannheim, Germany; galactose oxidase was
Glycoconjugate journal, 2016
The heparin disaccharides detected in farmed Atlantic salmon (Salmo salar) gills and intestines h... more The heparin disaccharides detected in farmed Atlantic salmon (Salmo salar) gills and intestines have, with one exception, been reported in porcine heparin. The relative amounts of disaccharides appear to be very different in the two species. Two chondroitin disaccharides with a proposed essential role in the zebrafish (Danio rerio) development and differentiation are detected in farmed Atlantic salmon. In addition, most of the chondroitin/dermatan sulfate and heparin disaccharides detected here have been reported in zebrafish, in support of the claims of the heparin presence in fish. The same chondroitin/dermatan disaccharides were detected in the bones of bony fishes. The rare disaccharide UA2S-GalNAc that was found in trace amounts in all 5 bony fishes was found in relative high amounts in gills and in significant amounts in intestines. The rare heparin disaccharide UA2S-GlcN was in relative highest amounts both in gills and intestines. In context with our previous reports, this c...
Bioorganic & medicinal chemistry letters, 2015
Pentapeptides have been shown to bind the synthetic heparin fondaparinux (Arixtra) as well the bi... more Pentapeptides have been shown to bind the synthetic heparin fondaparinux (Arixtra) as well the biological heparins dalteparin (Fragmin) and salmon heparin. In contrast to heparin binding consensus sequences, the pentapeptides are acidic or neutral, with no arginine or histidine residue. The peptides showed an effect on in vitro heparin anti-factor X activity with a reduction of fondaparinux activity by 65-95%. Heparin binding was further studied by using peptide solid phase chromatography and NMR analysis.
British Journal Of Nutrition
The in vitro digestion of caprine whey proteins was investigated by a two-step degradation assay,... more The in vitro digestion of caprine whey proteins was investigated by a two-step degradation assay, using human gastric juice (HGJ) at pH 2.5 and human duodenal juice (HDJ) at pH 7.5. Different protein and peptide profiles were observed after the first (HGJ) and second (HDJ) enzymatic degradation. The minor whey proteins serum albumin, lactoferrin and Ig were rapidly degraded by HGJ, while alpha-lactalbumin (alpha-LA) and beta-lactoglobulin (beta-LG) were more resistant and survived both 30 and 45 min of the enzymatic treatment. Further digestion with HDJ still showed intact beta-LG, and the main part of alpha-LA also remained unchanged. The protein degradation by HGJ and HDJ was also compared with treatment by commercial enzymes, by using pepsin at pH 2.5, and a mixture of trypsin and chymotrypsin at pH 7.5. The two methods resulted in different caprine protein and peptide profiles. The digests after treatment with HGJ and HDJ were screened for antibacterial effects on some selected ...
Biochemical Journal, 1974
1. The N-terminal fragment (PF-I) split off from prothrombin during coagulation was purified to h... more 1. The N-terminal fragment (PF-I) split off from prothrombin during coagulation was purified to homogeneity from human serum. 2. The apparent molecular weight is 27000±2000 in sodium dodecyl sulphate–polyacrylamide-gel electrophoresis, whereas a value of about 19600 is obtained by calculation based on amino acid and carbohydrate analyses. The N-terminal sequence is an Ala-Asx bond. The fragment contains about 16% carbohydrate, binds phospholipids in the presence of Ca2+and is adsorbed to BaSO4. The pKa of its BaSO4-binding group(s) is 3.1–3.5. 3. By CNBr cleavage of fragment PF-I two peptides (C-1 and C-2) were obtained with molecular weights of about 5900 (C-2) and 12400 (C-1) on the basis of amino acid and carbohydrate analyses. Only the smaller (N-terminal) peptide is adsorbed to BaSO4 and, since the ability of the whole protein to bind to BaSO4 is known to be absent in samples obtained from patients treated with vitamin K antagonists, this peptide probably contains the site of a...
Journal of Chromatography B, 2014
Protein Extraction from Green Plant Tissue
A method for preparation of protein from green plant tissue for two-dimensional electrophoresis i... more A method for preparation of protein from green plant tissue for two-dimensional electrophoresis is described. The method is demonstrated on barley leaves, potato leaves and spruce needles and appears to overcome the obstacles inherent in green plants to proteomic analysis. The yield and the representation of proteins are discussed.
Quantitation of Some Amino-Terminal Residues in Proteins Using 3H-LABELLED Dansyl Chloride and 14C-LABELLED Amino Acids
A method for quantitation of amino-terminal residues in proteins is presented. The method is a mo... more A method for quantitation of amino-terminal residues in proteins is presented. The method is a modification of a double isotope-labelling technique, using 3H-labelled dansyl chloride and 14C-labelled amino acids as internal standards. The method is demonstrated on human fibrinogen, horse myoglobin and on mouse myoloma IgA. A linear relationship between the ratio 3H/14C in the separated amino-terminal amino acid of the protein and the amount of protein added in the labelling mixture was obtained with standard deviations of +/- 7.4% +/- 3.4% and +/- 10.3%, respectively. An application of the method is demonstrated by measuring the increase in amino-terminal glycine in fibrinogen following the proteolytic action of thrombin. The method seems to be useful when 0.1 nmol or more of protein is used.
Journal of Dairy Science, 2013
Lactoferrin (LF) is a protein present in milk and other body fluids that plays important biologic... more Lactoferrin (LF) is a protein present in milk and other body fluids that plays important biological roles. As part of a diet, LF must survive gastrointestinal conditions or create bioactive fragments to exert its effects. The degradation of LF and formation of bioactive peptides is highly dependent on individual variation in intraluminal composition. The present study was designed to compare the degradation and peptide formation of bovine LF (bLF) following in vitro digestion under different simulated intraluminal conditions. Human gastrointestinal (GI) juices were used in a 2-step model digestion to mimic degradation in the stomach and duodenum. To account for variation in the buffering capacity of different lactoferrin-containing foods, gastric pH was adjusted either slowly or rapidly to 2.5 or 4.0. The results were compared with in vivo digestion of bLF performed in 2 volunteers. High concentration of GI juices and fast pH reduction to 2.5 resulted in complete degradation in the gastric step. More LF resisted gastric digestion when pH was slowly reduced to 2.5 or 4.0. Several peptides were identified; however, few matched with previously reported peptides from studies using nonhuman enzymes. Surprisingly, no bovine lactoferricin, f(17-41), was identified during in vitro or in vivo digestion under the intraluminal conditions used. The diversity of enzymes in human GI juices seems to affect the hydrolysis of bLF, generating different peptide fragments compared with those obtained when using only one or a few proteases of animal origin. Multiple sequence analysis of the identified peptides indicated a motif consisting of proline and neighboring hydrophobic residues that could restrict proteolytic process-ing. Further structure analysis showed that almost all proteolytic cutting sites were located on the surface and mainly on the nonglycosylated half of lactoferrin. Digestion of bLF by human enzymes may generate different peptides from those found when lactoferrin is digested by nonhuman enzymes. The degradation of LF in the GI tract should be taken into consideration when health effects are proposed, because LF has now been approved by the European Food Safety Authority as a dietary supplement in food products.
Thrombosis Research, 2010
Heparin was purified from gills and intestines from farmed Atlantic salmon (Salmo salar). Heparin... more Heparin was purified from gills and intestines from farmed Atlantic salmon (Salmo salar). Heparin activity was determined after size exclusion chromatography in the molecular weight range from above 8,000 to near 1,500. A specific activity of 110.1 antifactor Xa units/mg was measured in the less than 3,500 molecular weight fraction while 136.8 antifactor Xa units/mg was detected in a 8,000-3,500 molecular weight fraction. The presence of high affinity salmon heparin was demonstrated by using chromatography on antithrombin-Sepharose. Heparin with molecular weights lower than 3,500 was found both in high and low affinity fractions. NMR-analysis detected N-and O-sulfated oligosaccharides essential for heparin activity. The amount of salmon heparin with molecular weight lower than 8,000 varied from 12% to almost 100%. The factors determining this variation is not known, but appears to reside in the fish at the time of slaughter. The in vivo effect of salmon heparin was tested in rabbits using dalteparin as control. Salmon heparin activity was recovered in plasma samples expressed as antifactor Xa activity after intravenous administration. Based on a small number of samples and animals, the results indicate that in vivo half-life time of salmon heparin was higher than that of dalteparin.
European Journal of Biochemistry, 1977
European Journal of Biochemistry, 2000
Antithrombin, a major coagulation inhibitor in mammals, has for the first time been cDNA cloned f... more Antithrombin, a major coagulation inhibitor in mammals, has for the first time been cDNA cloned from a fish species. The predicted mature liver antithrombin of Atlantic salmon (Salmo salar) consists of 430 amino acids and shows about 67% sequence identity to mammalian and chicken antithrombins. Due to a single nucleotide replacement, Asn135 of the antithrombin in higher vertebrates is substituted by Asp in the salmon homolog. Hence, in contrast to the vertebrate antithrombins known so far, salmon antithrombin lacks the potential glycosylation site located close to the heparin binding site. The existence of only three N‐linked side chains is evidenced by the sequential removal of three carbohydrate chains from salmon antithrombin during timed‐digestion with N‐glycosidase F. The high heparin binding affinity of the salmon inhibitor, Kd of 2.2 and 48 nm at I = 0.15 and 0.3, respectively, is very similar to that of the minor human isoform β‐antithrombin, which is not glycosylated at Asn...
Physiologia Plantarum, 1993
The freezing resistance of the grass species Phleum pratense L. (timothy) and Phataris arundinace... more The freezing resistance of the grass species Phleum pratense L. (timothy) and Phataris arundinacea L. increases significantly after cold hardening. The content and composition of soluble carbohydrates were determined in the plants after short day treatment, cold hardening and dehardening. The amounts of mono-, di-and trisaccharides were reduced during the short day treatment, increased during cold hardening and decreased again during dehardening. The total amounts of soluble carbohydrates (mono-, di-, tri-and polysaccharides) were the same in hardened and dehardened plants, indicating that during hardening soluble polysaccharides (fructose polymers, fructans) were converted to mono-and oligosaccharides. Sucrose increased most after hardening conditions and, in P. arundinacea, a significant increase in 1-Ffructosylsucrose (isokestose) was also observed. Invertase (/3-fructofuranosidase, EC 3.2.1.26) activity increased following cold hardening and decreased following dehardening, while the a-galactosidase (EC 3.2.1.22) activity seemed to increase after dehardening. The glycosidases are probably involved in the mobilisation of polysaccharides during cold hardening.
Journal of Chromatography B, 2014
Heparin-binding proteins in human plasma were studied using affinity chromatography columns with ... more Heparin-binding proteins in human plasma were studied using affinity chromatography columns with porcine (2 mL, 10.7 mg capacity) and piscine heparin (5 mL, 2.7 mg capacity). Two-dimensional electrophoresis (Bio-Rad Protean II gel system with 16 cm × 16 cm gels using isoelectric focusing (IEF) and nonequilibrium pH-gradient gel electrophoresis (NEPHGE)), Bruker Ultraflex MALDI-TOF mass spectrometry and immunoblotting (NovaBlot semidry discontinuous blotting) were used for unfractionated plasma. This revealed electropherograms with differences between porcine and piscine heparin-binding and totally 17 different fibrinogen variants from all 3 chains. Immunodepletion was used to remove fibrinogen (42.1 mg anti-human fibrinogen in 8.4 mL resin) and serum albumin (0.42 mg binding capacity in 14 mL resin) and porcine and piscine heparin-binding proteins were identified using liquid chromatography-mass spectrometry (Ultimate 3000 NanoLC with Acclaim PepMap 100 column (50 cm × 75 m)-LTQ Orbitrap Mass XL). In total, the binding of 76 putative or acknowledged biomarkers are shown. Of the identified proteins, 14 are not previously shown to be heparin-binding, such as the low concentration proteins lipocalin-1 and tropomyosin and a hitherto not detected protein in plasma, zinc finger protein 483. The putative heparin-binding sequences were analyzed. The results suggest that the combination of group specific affinity and adapted immunodepletion chromatography could be useful in the study of the plasma proteome.
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Papers by Ragnar Flengsrud