Heparin-sepharose affinity

ApoE-containing HDL, obtained by heparin-Sepharose affinity chromatography, contains apoA-I, apoA-II, and apoC, as well as apoE (W12, W18). Clearance frou. the plasm, appears to be dependent on a specific hepatic receptor for apoE, which binds apoE-containing HDL and chylomicron remnants, but not other lipoproteins (H35, Mil, S28). Canine apoE-HDLC, with apoE as the only detectable apoprotein, is cleared from the plasma very rapidly by the liver (more than 90% in the first 20 minutes after intravenous injection) (M15). In adult man, dogs, and swine the apoE receptor numbers do not seem to be significantly reduced by cholesterol feeding (H35, Mil). 

T10. Trezzi, E., Calvi, C., Roma, P., and Catapano, A. L., Subfractionation of human very low density lipoproteins by heparin-Sepharose affinity chromatography. J. Lipid Res. 24, 790-795 (1983). 

W12. Weisgraber, K. H., and Mahley, R. W., Subfractionation of human high density lipoproteins by heparin-Sepharose affinity chromatography. /. Lipid Res. 21, 316-325 (1980). 

Although binding activity could be easily demonstrated in crude cell free mycelial extracts of phenylamide-sensitive Phytophthora strains, binding activity decreased considerably when the extracts were subjected to Heparin Sepharose affinity chromatography, a first step in the purification of RNA polymerases (16. Purification of the phenylamide receptor, therefore, cannot be followed using the binding assay. An alternative approach would be to covalently label the receptor with a photoaffinity probe. Purification of the labeled complex can then be followed either by a radioassay or an immunoassay, which respectively require a radiolabeled photoaffinity probe or antiserum that selectively recognizes covalently bound phenylamide residues. 

P. N. Srivastava and A. A. Farooqui. Heparin-Sepharose affinity chromatography for purification of bull seminal plasma hyaluronidase. Biochem. J. 755 531 (1979). 

Characterization of Displaced Protein. Two approaches were used simultaneously to characterize the I-thrombin displaced from heparin-PVA columns filtration on Sephadex G-200 and heparin-Sepharose affinity chromatography using the elution conditions described by Collen et al. (18) for the separation of antithrombin III from enzyme-antithrombin III complex. Radiolabelled antithrombin III displaced from the heparin-PVA column was characterized in a similar way by affinity chromatography on heparin-Sephrose. Detailed methods are presented elsewhere ( ... 

The importance of chemokines binding to endothelial surfaces was suggested soon after the family was identified by the demonstration that neutrophils migrated toward immobilized IL-8/CXCL8 by a mechanism called haptotaxis (Rot, 1993). Interestingly, the chemokine field actually started with the identification of a protein by heparin-sepharose affinity chromatography, platelet factor 4 (PF4)/CXCL4 (Deuel, Keim, Farmer, Heinrikson, 1977). 

Lipoprotein lipase (from bovine skimmed milk) [9004-02-8] [EC 3.1.1.34]. Purified by affinity chromatography on heparin-Sepharose [Shirai et al. Biochim Biophys Acta 665 504 1981]. 

A detailed examination of the affinity of SLPI for the heparinized capillary was next made using a stepwise elution (from 0.1 to 0.9 M NaCl) (Fig. 11). SLPI eluted from the capillary with 0.2 M NaCl. This agreed well with results obtained by traditional affinity chromatography on a heparin-Sepharose matrix. The ACE method has the unique advantages over traditional affinity chromatography in that it requires much smaller quantities of protein and afforded better separation profiles. 

Fig. 1. Chemokines have different relative affinities for heparin in an inunobilized-heparin competitive binding assay. Radiolabeled chemokines were incubated with either Heparin-Sepharose beads (O), Sepharose beads (A), or without beads ( ) in the presence of increasing concentrations of heparin. Competition assays with MCP-1 were performed as described in the Methods section. Each point represents the mean S.E.M. of triplicate determinations, and the experiment shown is a representative of 2-A experiments for each chemokine. The maximal binding was 3000-5000 cpm, which corresponded to 15-20% of the total added radioactivity.

Purify the lipase by affinity chromatography on heparin-Sepharose Ki 0.026mM for very low density lipoprotein. It is inhibited by 2-mercaptoethanol, Cys, Ca, Hg, Mg and Mn ions. Protamine sulfate, Img of bovine serum albumin/mL or in 50% glycerol at -70 , stabilises the lipase for several days. 60% loss of activity occurs at 0°/lhour in the presence of 1% of bovine serum albumin. [Shirai et al. Biochim Biophys Acta 665 504 7957.]... 

Lyon and Phelps [57] have evaluated various glycan-substituted AH-Sepharose gels to be used for affinity chromatography. Srivastava and Farooqui reported the elution of hyaluronidase from heparin-Sepharose columns using heparin or hyaluronan solutions as eluent [58]. Alternatively, affinity chromatography of hyaluronidase has been performed using concavalin A-Sepharose [58] or Matrex gel phenyl boronate [59] columns. 

As a further purification technique, affinity chromatography was explored. In preliminary experiments, a heparin-Sepharose column failed to bind heparinase. We therefore searched for a competitive and reversible heparinase inhibitor to act as a ligand. Three synthetic heparin substitutes— poly(vinyl sulfate), polyanethole sulfonate, and polystyrene sulfonate—met these requirements. The inhibitory effect of poly(vinyl sulfate) (K = 3.0 X 10 8M MW 10,000) appeared to be linked to the presence of sulfate groups because inhibition was lost when poly(vinyl sulfate) was hydrolyzed. 

Characterization of Displaced Protein. With labelled antithrombin III, chromatography of the displaced radioactivity on heparin-Sepharose revealed that the bulk of the displaced radioactive material did not bind to heparin-Sepharose (Table II). With arvinized plasma as the displacing eluent, 65% of the antithrombin III eluted in the void volume, compared with 49% of the control I-antithrombin III (diluted in citrated plasma) that had not previously been used to inactivate thrombin the latter unbound fraction was likely labelled impurities or inhibitor modified by radiolabelling to lose its heparin affinity. With 5% (w/v) albumin used as a displacing eluent, 78% of the I-antithrombin III came out in the void volume. This increase in material that did not bind to heparin after displacement from heparin-PVA was attributed to post-complex antithrombin III, a modification of the original inhibitor resulting from the inactivation of thrombin. Neither thrombin-antithrombin III complex nor free antithrombin III were detected in the 5% (w/v) albumin displaced fractions while there was a barely detectable amount of complex (6%) and free antithrombin III (4%) in the material displaced by arvinized plasma. With the control I-antithrombin III, 25% of the radioactivity was determined to be free antithrombin III and 2% as complex. The remainder (22-27%) was not recovered from the column. 

Analysis of the radioactivity displaced by arvinized plasma indicated the presence of thrombin-anti thrombin III complex (- 70%) and what was presumed to be thrombin-a-2-macroglobulin complex (" 30%). The bound thrombin is thought to react first with antithrombin III to produce a bound inactivated thrombin-anti thrombin III complex, which is dislodged from heparin by a yet unknown plasma component(s), decomplexed by an unknown mechanism to react with a-2-macroglobulin. This mechanism is illustrated in Figure 1. After displacement, the increase in I-antithrombin III which had lost its affinity for heparin-Sepharose was attributed to the production of a post complex antithrombin III on decomplexation of the inactive complex. This modified antithrombin III has been described by Lam et al. ( ), Fish et al. (25) and Marciniak (26). Neither free I-thrombin nor I-antithrombin III were detected in the displaced eluent. 

Bull seminal-plasm a hyaluronidase has been purified 180-fold by affinity chromatography on concanavalin A-Sepharose , heparin-Sepharose , and chromatography on Sephadex G-200 and Sephacryl S-200. With hyaluronic acid as the substrate, the specific activity and turnover number of purified hyaluronidase were 3.63 /Umol min per mg (10400 National Formulary units mg of protein) and 214min (mol of product formed per mol of enzyme per min), respectively. Polyacrylamide gel electrophoresis indicated that the purified enzyme migrated as a single band at pH 4.3 and 5.3. Bull seminal-plasma hyaluronidase was markedly inhibited by hydroxylamine, phenyl-hydrazine, and semicarbazide. 

We have been able to purify heat shock activator protein, a 110 kD polypeptide, to over 95% homogeneity from a 0.35 M NaCl nuclear extract prepared from heat shocked Drosophila tissue culture cells (3). The extract was chromatographed on heparin-Sepharose, twice sequentially on specific DNA-Sepharose (4 ml and 0.6 ml bed volumes), and on FPLC Mono S (a Pharmacia ion exchange matrix). A typical purification requires 1.5 x 10 cells from which several micrograms of homogenous heat shock activator protein are obtained. The yield at the affinity step is > 50%. Typical overall yields range between 10% and 20%. The... 

Heparin-Sepharose CL-6B for Affinity Chromatography Amersham Bioscience, Uppsala, Sweden. 

Anion-exchange HPLC (Mono Q HR 5/5 column) and affinity chromatography (Hi-Trap Heparin and Hi-Trap Blue-Sepharose columns) and IDA-ICP-MS with an octapole reaction system ( Se/ Se and °Sef Se)... 

Functions.—Heparin fractionated by gel filtration appeared to bind to two sites on antithrombin III (association constants 0.6 x 10 and 0.2 x 10 moll" ), whereas heparin prepared by affinity chromatography on matrix-bound antithrombin III appeared to bind to only one site (association constant 2.3 x 10 moll ). These results suggest that one of the binding sites on antithrombin III does not bind the most active heparin components, but accommodates heparin-like molecules which, although similar in size to the active heparin components, have little or no anticoagulant activity. Heparins with high or low affinities for antithrombin III exhibited no differences in their abilities to bind lipoprotein lipase. Studies of the interaction between the lipoprotein lipase from cow s milk and Sepharose-immobilized heparin have shown that heparin is poly-disperse. Whereas heparin facilitated complex formation between a-thrombin and antithrombin III, it had little effect on the interaction between p-thrombin and antithrombin III. ... 

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