Disaccharide analysis of chondroitin and heparin from farmed Atlantic salmon.

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 communication suggests that glycosaminoglycans in farmed Atlantic salmon heparin need further studies in order to clarify structure and function.

The binding of pentapeptides to biological and synthetic high affinity heparin.

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.

Affinity chromatography, two-dimensional electrophoresis, adapted immunodepletion and mass spectrometry used for detection of porcine and piscine heparin-binding human plasma proteins.

Heparin-binding proteins in human plasma were studied using affinity chromatography columns with porcine (2mL, 10.7mg capacity) and piscine heparin (5mL, 2.7mg capacity). Two-dimensional electrophoresis (Bio-Rad Protean II gel system with 16cm×16cm 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 (42.1mg anti-human fibrinogen in 8.4mL resin) and serum albumin (0.42mg binding capacity in 14mL resin) and porcine and piscine heparin-binding proteins were identified using liquid chromatography-mass spectrometry (Ultimate 3000 NanoLC with Acclaim PepMap 100 column (50cm×75µm)-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.

Antibacterial peptides derived from caprine whey proteins, by digestion with human gastrointestinal juice.

Peptides in caprine whey were identified after in vitro digestion with human gastrointestinal enzymes in order to determine their antibacterial effect. The digestion was performed in two continuing steps using human gastric juice (pH 2·5) and human duodenal juice (pH 8) at 37°C. After digestion the hydrolysate was fractionated and 106 peptides were identified. From these results, twenty-two peptides, located in the protein molecules, were synthesised and antibacterial activity examined. Strong activity of the hydrolysates was detected against Escherichia coli K12, Bacillus cereus RT INF01 and Listeria monocytogenes, less activity against Staphylococcus aureus ATCC 25 923 and no effect on Lactobacillus rhamnosus GG. The pure peptides showed less antibacterial effect than the hydrolysates. When comparing the peptide sequences from human gastrointestinal enzymes with previously identified peptides from non-human enzymes, only two peptides, ß-lactoglobulin f(92-100) and ß-casein f(191-205) matched. No peptides corresponded to the antibacterial caprine lactoferricin f(14-42) or lactoferrampin C f(268-284). Human gastrointestinal enzymes seem to be more complex and have different cleavage points in their protein chains compared with purified non-human enzymes. Multiple sequence alignment of nineteen peptides showed proline-rich sequences, neighbouring leucines, resulting in a consensus sequence LTPVPELK. In such a way proline and leucine may restrict further proteolytic processing. The present study showed that human gastrointestinal enzymes generated different peptides from caprine whey compared with non-human enzymes and a stronger antibacterial effect of the hydrolysates than the pure peptides was shown. Antimicrobial activity against pathogens but not against probiotics indicate a possible host-protective activity of whey.

Purification, characterization and in vivo studies of salmon heparin.

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.

Structural characteristic, pH and thermal stabilities of apo and holo forms of caprine and bovine lactoferrins.

Apo and holo forms of lactoferrin (LF) from caprine and bovine species have been characterized and compared with regard to the structural stability determined by thermal denaturation temperature values (T (m)), at pH 2.0-8.0. The bovine lactoferrin (bLF) showed highest thermal stability with a T (m) of 90 ± 1°C at pH 7.0 whereas caprine lactoferrin (cLF) showed a lower T (m) value 68 ± 1°C. The holo form was much more stable than the apo form for the bLF as compared to cLF. When pH was gradually reduced to 3.0, the T (m) values of both holo bLF and holo cLF were reduced showing T (m) values of 49 ± 1 and 40 ± 1°C, respectively. Both apo and holo forms of cLF and bLF were found to be most stable at pH 7.0. A significant loss in the iron content of both holo and apo forms of the cLF and bLF was observed when pH was decreased from 7.0 to 2.0. At the same time a gradual unfolding of the apo and holo forms of both cLF and bLF was shown by maximum exposure of hydrophobic regions at pH 3.0. This was supported with a loss in α-helix structure together with an increase in the content of unordered (aperiodic) structure, while ß structure seemed unchanged at all pH values. Since LF is used today as fortifier in many products, like infant formulas and exerts many biological functions in human, the structural changes, iron binding and release affected by pH and thermal denaturation temperature are important factors to be clarified for more than the bovine species.

Protein extraction from green plant tissue.

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.

In vitro studies of the digestion of caprine whey proteins by human gastric and duodenal juice and the effects on selected microorganisms.

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 microorganisms, Escherichia coli, Bacillus cereus, Lactobacillus rhamnosus GG and Streptococcus mutans. Active growing cells of E. coli were inhibited by the digestion products from caprine whey obtained after treatment with HGJ and HDJ. Cells of B. cereus were inhibited only by whey proteins obtained after reaction with HGJ, while the products after further degradation with HDJ demonstrated no significant effect. Screenings performed on cells of Lb. rhamnosus GG and S. mutans all showed no signs of inhibition.

Sample displacement chromatography of Atlantic Salmon (Salmo salar) thrombin.

A modified method of sample displacement chromatography (SDC) was used to purify active salmon thrombin on a heparin-coupled matrix to near homogeneity in milligram amounts from 117 ml plasma. This was achieved by combining a low-pressure multi-column affinity chromatography system with non-homogenous sample application in the order of increasing affinity to Heparin Sepharose. The results suggest that this modified method could be useful in protein purification. Some characteristics of salmon thrombin are presented.

Purification and characterization of Atlantic salmon (Salmo salar) fibrinogen.

This study describes a purification protocol of salmon fibrinogen that gives a consumable and highly clottable fibrinogen. Some characteristics of salmon and human fibrinogen are compared. Fibrinogen was purified from barium sulphate adsorbed plasma of Atlantic salmon, using two steps of 25% ammonium sulphate precipitation followed by ultrafiltration. The clottability of the purified salmon fibrinogen was 91%. The Aalpha chains of salmon fibrinogen were heterogeneous with a molecular mass of 90-110 kDa, compared to approximately 67 kDa of human fibrinogen Aalpha chains. The Bbeta and gamma chains of salmon and human fibrinogen had molecular masses of approximately 55 and 50 kDa, respectively. Western blotting revealed that polyclonal rabbit anti-human fibrinogen antibodies had affinity for the gamma chains of salmon fibrinogen, making it possible to study factor XIII activity in purified salmon fibrinogen. Cross-linking of either gamma-gamma or gamma-alpha chains was not detected upon incubation of the purified fibrinogen with thrombin and calcium alone, but was detected when clotting was performed in plasma indicating absence of factor XIII activity in the purified product.