Phosphotransbutyrylase from Clostridium acetobutylicum ATCC 824 and its role in acidogenesis.

Phosphotransbutyrylase (phosphate butyryltransferase [EC 2.3.1.19]) from Clostridium acetobutylicum ATCC 824 was purified approximately 200-fold to homogeneity with a yield of 13%. Steps used in the purification procedure were fractional precipitation with (NH4)2SO4, Phenyl Sepharose CL-4B chromatography, DEAE-Sephacel chromatography, high-pressure liquid chromatography with an anion-exchange column, and high-pressure liquid chromatography with a hydrophobic-interaction column. Gel filtration and denaturing gel electrophoresis data were consistent with a native enzyme having eight 31,000-molecular-weight subunits. Within the physiological range of pH 5.5 to 7, the enzyme was very sensitive to pH change in the butyryl phosphate-forming direction and showed virtually no activity below pH 6. This finding indicates that a change in internal pH may be one important factor in the regulation of the enzyme. The enzyme was less sensitive to pH change in the reverse direction. The enzyme could use a number of substrates in addition to butyryl coenzyme A (butyryl-CoA) but had the highest relative activity with butyryl-CoA, isovaleryl-CoA, and valeryl-CoA. The Km values at 30 degrees C and pH 8.0 for butyryl-CoA, phosphate, butyryl phosphate, and CoASH (reduced form of CoA) were 0.11, 14, 0.26, and 0.077 mM, respectively. Results of product inhibition studies were consistent with a random Bi Bi binding mechanism in which phosphate binds at more than one site.     

Bile acids LIII: Application of reverse-phase high-pressure liquid chromatography to the analysis of conjugated bile acids in bile samples

The common conjugated bile acids of deproteinated bile from the human or the rat can be separated by high-pressure liquid chromatography and quantitated within 30 min with a 4-mm × 30-cm “fatty-acid analysis” column (Waters Associates) in 2-propanol/8.8 mm potassium phosphate buffer (pH 2.5) 160:340, coupled to a uv flow detector set at 193-nm wavelength. Detection limits were at least 0.1 nmol for the tauro-conjugates and 0.2 nmol for the glyco-derivatives.     

Purification and properties of an acetoacetyl coenzyme A-reacting phosphotransbutyrylase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593

During the study of acetoacetyl coenzyme A (CoA)-reacting enzymes of Clostridium beijerinckii NRRL B593, a phosphate-dependent acetoacetyl-CoA-utilizing activity was detected in protein fractions devoid of thiolase and phosphotransacetylase. Further purification of this acetoacetyl-CoA-utilizing activity yielded an enzyme which may be designated as phosphotransbutyrylase (PTB; phosphate butyryltransferase [EC 2.3.1.19]). PTB from C. beijerinckii NRRL B593 was purified 160-fold with a yield of 14% and, with the best fractions, purified 190-fold to near homogeneity. It showed a native Mr of 205,000 and a subunit Mr of 33,000. PTB activity was sensitive to pH changes within the physiological range of 6 to 8. PTB exhibited a broad substrate specificity. The Km values at pH 7.5 for butyryl-CoA, acetoacetyl-CoA, and acetyl-CoA were 0.04, 1.10, and 3.33 mM, respectively. The Vmax values with butyryl-CoA and acetoacetyl-CoA were comparable, but the Vmax/Km was higher for butyryl-CoA than for acetoacetyl-CoA. An apparent Km of 6.5 mM for phosphate was obtained with butyryl-CoA as the cosubstrate, whereas it was 12.9 mM with acetoacetyl-CoA as the cosubstrate. It remains to be established whether the putative compound acetoacetyl phosphate is produced in the PTB-catalyzed reaction with acetoacetyl-CoA.     

Reverse-phase high-pressure liquid chromatography of uridine diphosphate N-acetylmuramyl peptide precursors of bacterial cell wall peptidoglycan

A series of seven different UDP-N-acetylmuramyl peptide precursors of bacterial cell wall peptidoglycan was examined by reverse-phase high-pressure liquid chromatography. Mixtures of these compounds were successfully and rapidly analyzed by using a Waters μBondapak C₁₈ column as a stationary phase and isocratic elutions with 0.05 m ammonium phosphate or formate buffers of appopriate pH. Furthermore, their accurate quantitation could also be readily achieved by reverse-phase high-pressure liquid chromatography. All these techniques should be extremely useful for the purification of these compounds and for a wide range of biochemical studies concerning the cytoplasmic steps of the biosynthesis of peptidoglycan.     

A time-resolved assay of dopamine β-hydroxylase activity utilizing high-pressure liquid chromatography

A time-resolved assay of dopamine β-hydroxylase (EC 1.14.17.1) activity utilizing high-pressure liquid chromatography is described. The conversion of tyramine to octopamine by the enzyme was used as a standard reaction. The analytical separation of the assay substrate and product employed a reversed-phase ion-pair chromatographic system, with ultraviolet absorbance detection of eluents at 280 nm. Aliquots of the assay solution were injected directly onto the high-pressure liquid chromatography column and were separated in 6 min total elapsed time, thus permitting time-resolved determination of the produet. Quantities of octopamine as small as 20 pmol could be measured. This facile method is more straightforward, convenient, and sensitive than previously published physical and spectroscopic methods of determining dopamine β-hydroxylase activity.     

Oxidation-reduction potentials of butyryl-CoA dehydrogenase

In order to obtain butyryl-CoA dehydrogenase from Megasphaera elsdenii in pure enough form to perform redox studies, the existing purification procedures first had to be modified and clarified [Engel, P. (1981) Methods Enzymol. 71, 359-366]. These modifications are described, and the previously unpublished spectral properties of the electrophoretically pure CoA-free butyryl-CoA dehydrogenase are presented. In our spectral reductive titration of pure enzyme, we show that although blue neutral flavin radical is stabilized in nonquantitative amounts in dithionite titrations (19%) or in electrochemical reductions mediated by methylviologen (5%), it is not thermodynamically stabilized; therefore, only a midpoint potential for butyryl-CoA dehydrogenase is obtained. The electron-transfer behavior from pH 5.5 to pH 7.0 indicates reversible two-electron transfer accompanied by one proton: EFlox + 2e- + H+ = EFlredH- Em7 = -0.079 V vs. SHE where EFlox is oxidized butyryl-CoA dehydrogenase, EFlredH- is two electron reduced enzyme, and Em7 is the midpoint potential at pH 7.0 at 25 degrees C. Redox data and activity data both indicate that the enzyme loses activity rapidly at pH values above 7.0. The Em7 of the butyryl-CoA dehydrogenase is 40 mV positive of the Em7 of the butyryl-CoA/crotonyl-CoA couple [Gustafson, W. G., Feinberg, B. A., & McFarland, J. T. (1986) J. Biol. Chem. 261, 7733-7741]. Binding of substrate analogue acetoacetyl-CoA caused the potential of butyryl-CoA dehydrogenase to shift 100 mV negative of the free enzyme. The negative shift in potential makes electron transfer from enzyme to substrate more probable, which is consistent with the direction of electron transfer in the bacterial system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bioconversion of leukotriene D4 by lung dipeptidase

Sheep lung dipeptidase was released from a lung membrane preparation by digestion with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. The total enzyme activity released into the supernatant was 4- to 5-fold greater than that measured in the intact membrane prior to solubilization. The release of the peptidase from the membrane by this treatment is typical of proteins anchored to the lipid bilayer by a covalent attachment of phosphatidylinositol via a C-terminal glycolipid extension. The solubilized lung peptidase was further purified by ammonium sulfate fractionation followed by affinity chromatography and high-pressure liquid chromatography. A linear relationship between log molecular weight and elution volume for proteins of known molecular weight was established using a Toya Soda TSK 3000 high-pressure liquid chromatography column, and the molecular weight of the lung dipeptidase was estimated at 105,000. The peptidase activity against glycyldehydrophenylalanine of the purified enzyme co-chromatographed in high-pressure liquid chromatography with the activity that converted leukotriene D4 to leukotriene E4. In kinetic studies using leukotriene D4 as substrate, the relationship between the rate of hydrolysis and enzyme concentration was shown to be linear over the range 20 ng to 98 ng enzyme. Values of Km and Vmax for the dipeptidase using leukotriene D4 as substrate were 43 +/- 6 microM and 11,200 +/- 400 nmol/min per mg, respectively. Inhibition of the conversion of leukotriene D4 to leukotriene E4 was observed with a series of inhibitory agents. Cilastatin, bestatin and chloracetyldehydrophenylalanine were all effective at the micromolar level with cilastatin proving to be the most effective inhibitor. Dithiothreitol was effective within the millimolar range.     

Preparation of homogenous NADPH cytochrome c (P-450) reductase from house flies using affinity chromatography techniques.

NADPH-cytochrome c (P-450) reductase (EC 1.6.2.4) was purified to apparent homogeneity from microsomes of house flies, Musca domestica L. The purification procedure involves column chromatography on three different resins. The key step in the purification scheme is the chromatography of the enzyme mixture on an affinity column of agarose-hexane-nicotinamide adenine dinucleotide phosphate. The enzyme has an estimated molecular weight of 83,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contains 1 mol each of FAD and FMN per mol of enzyme. The enzyme exhibited a Bi Bi ping-pong kinetic mechanism with NADPH and cytochrome c. The Vmax and Km for cytochrome c were 42.3 mumol min-1 mg-1 and 12.7 muM, respectively. Turnover numbers based on micromoles of enzyme were 2,600 min-1. NADP+ and 2'-AMP both inhibited the reductases with apparent Ki values of 6.9 and 187 muM, respectively. These preparations of NADPH-cytochrome c reductase were found to reduce purified house fly cytochrome P-450 in the presence of NADPH.     

Purification and characterization of 3-hydroxyisobutyrate dehydrogenase from rabbit liver

3-Hydroxyisobutyrate dehydrogenase (3-hydroxy-2-methyl propanoate: NAD+ oxidoreductase, EC 1.1.1.31) was purified 1800-fold from rabbit liver by detergent extraction, differential solubility in polyethylene glycol and (NH4)2SO4, and column chromatography on DEAE-Sephacel, phenyl-Sepharose, CM(carboxymethyl)-Sepharose, Affi-Gel Blue, and Ultrogel AcA-34. The enzyme had a native Mr of 74,000 and appeared to be a homodimer with subunit Mr = 34,000. The enzyme was specific for NAD+. It oxidized both S-3-hydroxyisobutyrate and R-3-hydroxyisobutyrate, but the kcat/Km was approximately 350-fold higher for the S-isomer. Steady state kinetic analysis indicates an ordered Bi Bi reaction mechanism with NAD+ binding before 3-hydroxyisobutyrate. The enzyme catalyzed oxidation of S-3-hydroxyisobutyrate between pH 7.0 and 11.5 with optimal activity between pH 9.0 and 11.0. The enzyme apparently does not have a metal ion requirement. Essential sulfhydryl groups may be present at both the 3-hydroxyisobutyrate and NAD+ binding sites since inhibition by sulfhydryl-binding agents was differentially blocked by each substrate. The enzyme is highly sensitive to product inhibition by NADH which may play an important physiological role in regulating the complete oxidation of valine beyond the formation of 3-hydroxyisobutyrate.     

Isolation and properties of rat plasma lecithin-cholesterol acyltransferase

Lecithin-cholesterol acyltransferase was purified from rat plasma and the properties of this enzyme during the purification procedures and those of the purified enzyme were investigated in comparison with the human enzyme. The rat enzyme was not adsorbed on hydroxyapatite, which was employed for the purification of the human enzyme. When purified human enzyme was incubated at 37 degrees C in 0.1 mM phosphate buffer (pH 7.4; ionic strength, 0.00025), no alteration of enzyme activity was observed for up to 6 h. In the case of the rat enzyme, however, approximately 40% of the enzyme activity was lost under the same conditions. The human enzyme and rat enzyme were both retained on a Sepharose 4B column to which HDL3 was covalently linked, in 39 mM phosphate buffer, pH 7.4. Although the human enzyme was eluted from the column in 1 mM phosphate buffer, the rat enzyme was dissociated from the column at a lower buffer concentration (0.1 mM phosphate buffer). These findings indicate that the rat enzyme effectively associated with HDL3 in 39 mM phosphate buffer, pH 7.4, but the association was more sensitive to increase of ionic strength compared with that of the human enzyme.     

Studies on phosphoribosylpyrophosphate synthetase from Giardia intestinalis.

A substantial degree of purification, up to 3200-fold, with recoveries of 8-11% of phosphoribosylpyrophosphate (P-Rib-PP) synthetase from Giardia intestinalis extracts was achieved by the high resolution techniques of anion exchange chromatography and chromatofocusing columns on a fast protein liquid chromatography system. A Mono P chromatofocusing column gave rise to an enzyme peak eluting from the column at pH 4.5, indicating that the enzyme has an isoelectric point (pI) at approximately this value. The molecular weight of the enzyme was found to be 150,000 from a Sephacryl S-200 column. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified enzyme gave a single protein band with a subunit molecular weight of 38,000, indicating that the enzyme existed as a tetramer. The properties of G. intestinalis P-Rib-PP synthetase in terms of pH optimum, isoelectric point, subunit structure, phosphate requirement, metal and nucleotide specificity, appear to be very similar to those of the enzyme from other sources.     

Analysis of oligosaccharides from heparin by reversed-phase ion-pairing high-performance liquid chromatography

An ion-pairing high-pressure liquid chromatography procedure was developed for analysis of mixtures of oligosaccharides generated by nitrous acid cleavage of heparin. Oligosaccharides were eluted from a Hi-Chrom 5S ODS (C18) column using mixtures of acetonitrile and buffers containing 40 mM ammonium phosphate and 1 mM tetrabutylammonium phosphate. Isocratic conditions were developed for optimal separation of a number of individual disaccharides and tetrasaccharides that were characterized previously (M.J. Bienkowski and H.E. Conrad (1985) J. Biol. Chem. 260, 356-365). These isocratic conditions were then coupled to obtain gradient elution conditions for the ion-pairing separations of mixtures of disaccharides and mixtures of tetrasaccharides. A comparison of the elution profiles obtained in the ion-pairing chromatography procedure with profiles obtained by anion-exchange high-pressure liquid chromatography profiles showed markedly better overall resolution by the ion-pairing procedure. As a result of this improved resolution, the new procedure showed the presence of previously unidentified products in the heparin oligosaccharide mixtures.     

Affinity chromatography of amine oxidase from Aspergillus niger.

Omega-Aminohexyl-Sepharose 4B served as an excellent biospecific adsorbent for affinity chromatography of amine oxidase (monoamine:O2 oxidoreductase (deaminating), EC 1.4.3.4) from Aspergillus niger. The enzyme was completely adsorbed on this affinity resin when applied to a column in 0.1 M potassium phosphate buffer (pH 7.2). Although a small part of the enzyme was retained on the column through ionic interaction and eluted with 1.0 M potassium phosphate buffer (pH 7.2), most of the enzyme adsorbed was eluted with 0.5 M potassium phosphate buffer (pH 7.2) containing 10 mM butylamine. Essentially no retention of the enzyme on a column of epsilon-aminopentyl-Sepharose or delta-aminobutyl-Sepharose occurred under the same conditions, indicating that an appropriate length (more than approx. 12 A) of a hydrocarbon extension between the agarose matrix and the terminal amino group would be necessary for efficient adsorption of amine oxidase. The modification of the enzyme with 3-methyl-2-benzothiazolinone hydrazone (carbonyl inhibitor) or dithionite (reducing agent) resulted in loss of the ability to bind to omega-aminohexyl-Sepharose. It was also demonstrated that the affinity chromatography on omega-aminohexyl-Sepharose can be used as a powerful means of purifying this enzyme from crude extracts of Aspergillus niger. All of the three adsorbents were effective as a substrate in the amine oxidase reaction, but their substrate activities were as low as the corresponding free diamines.     

Human placental sialidase: partial purification and characterization

A sialidase [EC 3.2.1.18] has been partially purified from human placenta by means of procedures comprising Con A-Sepharose adsorption, ammonium sulfate precipitation, sucrose density gradient centrifugation, and high-pressure liquid chromatography on a Shim pack Diol 300 column. On high-pressure liquid chromatography, most of the beta-galactosidase that comigrated with the sialidase on sucrose density gradient centrifugation was removed. The sialidase was purified 3,600-fold from the preparation obtained by Con A-Sepharose adsorption. The enzyme liberated the sialic acid residues from (alpha 2-3) and (alpha 2-6) sialyllactose, colomic acid, fetuin, and transferrin, but not from bovine submaxillary mucin. The enzyme also hydrolyzed gangliosides GM3, GD1a, and GD1b in the presence of sodium cholate as a detergent, but GM1 and GM2 were less susceptible to the enzyme. The optimum pHs for 4-methylumbelliferyl-N-acetylneuraminate, sialyllactose, fetuin, and GM3 lay between 4.0 and 5.0.     

Purification of tyrosine hydroxylase by high-pressure liquid chromatography

Our study of tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) from rabbit adrenals has identified two major requirements which are likely to be of general application for the optimal purification and recovery of enzyme activity consequent to high-pressure liquid chromatography: (i) recovery of activity is maximized by pretreatment of the high-pressure liquid chromatography column before each use with protein to saturate high affinity, nonspecific sites exposed by the methanol used for washing, and storage of the column. (ii) Both purification and recovery are critically dependent upon the molarity of the mobile phase buffer. Examination of high-pressure liquid chromatography purified rabbit adrenal tyrosine hydroxylase by nondenaturing gel electrophoresis indicated that tyrosine hydroxylase activity was associated with one of the two protein bands in the gel. Thus, the convenient purification procedure described in this report leads to preparative amounts of tyrosine hydroxylase which is approximately 50% homogeneous.     

High performance liquid chromatography studies on protein: multiple forms of soybean lipoxygenase-1.

The multiplicity of components of soybean lipoxygenase-1 (EC 1.13.11.12) was shown by high performance liquid chromatography on the cation-exchange resin, TSK Gel LS-212 eluted with sodium phosphate at pH 6.8. Each component of the enzyme also showed a different chromatogram on the TSK Gel W type column, with which the separation is determined mainly by molecular size. However, the enzymes separated by chromatography on TSK Gel LS-212 have similar characteristics (pH optimum, heat stability, and inactivation by H₂O₂) and similar amino acid composition. The merits and limitations of high performance liquid chromatography in its application to analysis of proteins are discussed.     

Affinity chromatography of amine oxidase from Aspergillus niger

ω-Aminohexyl-Sepharose 4B served as an excellent biospecific adsorbent for affinity chromatography of amine oxidase (monoamine:O₂ oxidoreductase (deaminating), EC 1.4.3.4) from Aspergillus niger. The enzyme was completely adsorbed on this affinity resin when applied to a column in 0.1 M potassium phosphate buffer (pH 7.2). Although a small part of the enzyme was retained on the column through ionic interaction and eluted with 1.0 M potassium phosphate buffer (pH 7.2), most of the enzyme adsorbed was eluted with 0.5 M potassium phosphate buffer (pH 7.2) containing 10 mM butylamine. Essentially no retention of the enzyme on a column of ε-aminopentyl-Sepharose or δ-aminobutyl-Sepharose occurred under the same conditions, indicating that an appropriate length (more than approx. 12 Å) of a hydrocarbon extension between the agarose matrix and the terminal amino group would be necessary for efficient adsorption of amine oxidase. The modification of the enzyme with 3-methyl-2-benzothiazolinone hydrazone (carbonyl inhibitor) or dithionite (reducing agent) resulted in loss of the ability to bind to ω-aminohexyl-Sepharose. I was also demonstrated that the affinity chromatography on ω-aminohexyl-Sepharose can be used as a powerful means of purifying this enzyme from crude extracts of Aspergillus niger. All of the three adsorbents were effective as a substrate in the amine oxidase reaction, but their substrate activities were as low as the corresponding free diamines.     

Affinity-chromatography purification of alkaline phosphatase from calf intestine.

A crude preparation of alkaline phosphatase (EC 3.1.3.1) from calf intestinal mucosa was purified by affinity chromatography on Sepharose-bound derivatives of arsanilic acid, which was found to be a competitive inhibitor of the enzyme. Three biospecific adsorbents were prepared for the chromatography, and the best results were obtained with a tyraminyl-Sepharose derivative coupled with the diazonium salt derived from 4-(p-aminophenylazo)phenylarsonic acid. Alkaline phosphatase was the only enzyme retained by the affinity column in the absence of Pi. The enzyme eluted by phosphate buffer had a specific activity of about 1200 units per mg of protein at pH 10.0, with 5.5mM-p-nitrophenyl phosphate as the substrate.     

Large-scale preparation and reconstitution of apo-flavoproteins with special reference to butyryl-CoA dehydrogenase from Megasphaera elsdenii. Hydrophobic-interaction chromatography

A new method is described for the large-scale reversible dissociation of flavoproteins into apoprotein and prosthetic group using hydrophobic-interaction chromatography. Lipoamide dehydrogenase from Azotobacter vinelandii and butyryl-CoA dehydrogenase from Megasphaera elsdenii are selected to demonstrate the usefulness of the method. In contrast to conventional methods, homogeneous preparations of apoproteins in high yields are obtained. The apoproteins show high reconstitutability. The holoenzymes are bound to phenyl-Sepharose CL-4B at neutral pH in the presence of ammonium sulfate. FAD is subsequently removed at pH 3.5-4.0 by addition of high concentrations of KBr. Large amounts of apoenzymes (200-500 mg), showing negligible residual activity, are eluted at neutral pH in the presence of 50% ethylene glycol. The holoenzyme of lipoamide dehydrogenase can be reconstituted while the apoprotein is still bound to the column or the apoenzyme can be isolated in the free state. In both cases the yield and degree of reconstitution of holoenzyme is more than 90% of starting material. Apo-lipoamide-dehydrogenase exists mainly as a monomer in solution and reassociates to the native dimeric structure in the presence of FAD. The apoenzyme is stable for a long period of time when kept in 50% ethylene glycol at -18 degrees C. Steady-state fluorescence-polarization measurements of protein-bound FAD indicate that reconstituted lipoamide dehydrogenase possesses a high stability which is governed by the low dissociation rate constant of the apoenzyme-FAD complex. The holoenzyme of butyryl-CoA dehydrogenase cannot be reconstituted when the apoenzyme is bound to the column. However, stable apoprotein can be isolated in the free state yielding 50-80% of starting material, depending on the immobilization conditions. The coenzyme A ligand present in native holoenzyme is removed during apoprotein preparation. The apoenzyme is relatively stable when kept in 50% ethylene glycol at -18 degrees C. From kinetic and gel filtration experiments it is concluded that the reconstitution reaction of butyryl-CoA dehydrogenase is governed by both the pH-dependent hydrodynamic properties of apoenzyme and the pH-dependent stability of reconstituted enzyme. At pH 7, the apoenzyme is in equilibrium between dimeric and tetrameric forms and reassociates to a native-like tetrameric structure in the presence of FAD. The stability of reconstituted enzyme is strongly influenced by the presence of CoA ligands as shown by fluorescence-polarization measurements. The degree of reconstitution of butyryl-CoA dehydrogenase is more than 80% of the original specific activity under certain conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification of chondroitinase B and chondroitinase C using glycosaminoglycan-bound AH-Sepharose 4B

Chondroitinase B and chondroitinase C were separated from an extract of Flavobacterium heparinum induced with chondroitin 6-sulfate by using column chromatography on hydroxylapatite. Chondroitinase C was eluted together with the activities of hyaluronidase, delta4,5glycosiduronase, and sulfatase. The latter two activities were eliminated exclusively by passing the crude chondroitinase C fraction through a phosphono-cellulose column pre-equilibrated with 0.07M sodium phosphate buffer (pH 6.8). Chondroitinase C was then purified by affinity chromatography using dermatan sulfate-bound AH-Sepharose 4B coated with the same glycosaminoglycan. Purification of the enzyme was achieved 18-fold and in 73% yield. On the other hand, the activities of delta4,5glycosiduronase and sulfatase were decreased to 50 and 60%, respectively, as compared with those in the crude chondroitinase B fraction, after passing the fraction through a column of phosphono-cellulose pre-equilibrated with 0.1M sodium phosphate buffer (pH 6.8). The remaining activities of these two enzymes were then eliminated from chondroitinase B by affinity chromatography with heparin-bound AH-Sepharose 4B coated with dermatan sulfate. In the affinity chromatography used in the present study, non-covalent coating of the glycosaminoglycan-bound (covalently) AH-Sepharose 4B with the same or another glycosaminoglycan was found to be important.