Robert E. Hurst

Biochemical composition and heterogeneity of heparan sulfates isolated from AH-130 ascites hepatoma cells and fluid

The glycosaminoglycan composition of AH-130 ascites hepatoma cells and fluid were examined using enzymatic digestion, electrophoresis, and sequential partition fractionation. The cell-associated glycosaminoglycans were found to consist of 93% heparan sulfate, with the remainder consisting primarily of chondroitin sulfate. The glycosaminoglycans isolated from the ascitic fluid were found to consist of 58% heparan sulfate, 26% hyaluronic acid and 16% chondroitin sulfate. Dermatan sulfate was not detected in either cells or fluid. The heparan sulfate isolated from AH-130 cells in low-sulfate and highly heterogeneous with respect to biochemical composition. Fractions isolated by partition fractionation varied from 0.14 mol sulfate/mol uronic acid to 0.6 mol sulfate/mol uronic acid. Of the total sulfate 70--80% is N-sulfate in the former and 50% in the latter. Electrophoresis in 0.1 M HCl showed a highly heterogeneous material with mobility between that of hyaluronic acid and beef lung heparan sulfate. The heparan sulfate isolated from the fluid was similar to that isolated from the cells but was, however, somewhat more homogeneous with respect to charge.

A method for the quantitative determination of urinary glycosaminoglycans

A method is presented by which the urinary glycosaminoglycans can be isolated in a macromolecular fraction with complete recovery by preparative gel filtration. The urinary glycosaminoglycans can be quantitated by determinations of uronic acid and sulfaminohexose in the macromolecular fraction. Using untreated, random urine specimens of less than 5 ml volume, clinical mucopolysaccharidoses types I, II, and III could be readily distinguished.

An accurate colorimetric method for measurement of sulfaminohexose in heparins and heparan sulfates

Abstract A modification of a method for hexosamine analysis is presented which adapts it to measurement of sulfaminohexose in heparins and heparan sulfates. Unlike methods of sulfaminohexose analysis based upon coupling with indole, the absorptivity of polymeric and monomeric hexosamines is identical. N -Sulfated hexosamines are specifically deaminated in 33% acetic acid to yield free 2,5-anhydromannose residues which are then coupled to the color reagent 3-methyl-2-benzothiazolinone hydrazone hydrochloride. The sulfaminohexose content of a variety of heparins and heparan sulfates was determined with this methodology and compared with the indole-coupling method. Interferences by amino acids, proteins, and neutral sugar were evaluated in the sulfaminohexose assay and in the originally reported procedure for total hexosamine analysis.

Partition techniques for isolation and fractionation of urinary glycosaminoglycans

Abstract A method for the isolation and a second method for the fractionation of urinary glycosaminoglycans (GAG) are presented. Both methods are based upon the partition of complexes formed between GAG and quaternary ammonium salts in two-phase systems of butanol and aqueous salt. The method for isolation permits the complete recovery, in one step, of the total urinary GAG in high purity. The other method presents a technique by which urinary GAG can be fractionated with high resolution. Each of the mucopolysaccharidosis types examined afforded a distinctive fractionation pattern, and several structural differences in the GAG excreted in these disorders were noted.

The partition behavior of complexes of glycosaminoglycans and quaternary ammonium salts

Complexes of purified glycosaminoglycans and hexadecylpyridinium chloride are shown to be capable of partition between aqueous and butanol phases. The partition coefficient of these complexes is dependent upon the concentration of the supporting electrolyte as well as the concentration of the quaternary ammonium salt. A sharp transition, during which the complex changes from complete solubility in the butanol phase to complete solubility in the aqueous phase, occurs over a very narrow range of salt concentrations. The various glycosaminoglycans show differences sufficient to permit fractionation at least into nonsulfated, monosulfate, and polysulfated classes by simple partition.

The partition of glycosaminoglycan-quarternary ammonium complexes II. The effects of polymer molecular weight and sulfation

Previous results have shown the possibility for obtaining high-resolution separations of glycosaminoglycans by partition in butanol/aqueous two-phase systems containing quaternary ammonium salts. In this paper, the effects on partition behavior of both polymer molecular weight and sulfation were examined. Two series of fractionated chondroitin sulfate polymers were isolated in which the molecular weight and sulfation varied systematically. In the molecular weight series the six samples, spanned the range from 3200 +/- 300 to 19 700 +/- 500 and each sample carried 0.8 sulfate groups per uronic acid residue. In the sulfation series, each sample had an essentially constant molecular weight of 13 000, but the sulfation varied from 0.58 to 0.88 sulfate groups per uronic acid. The C50 of each of these samples was determined in the 1-butanol/aqueous NaCl phase system containing 1% hexadecylpyridinium chloride. In the series wherein the molecular weight varied, the C50 increased with molecular weight up to 12 000 where a limiting value was reached. In the series wherein the sulfation varied, a linear relationship was found between the C50 and the square of the number of anionic substituents per disaccharide. These results show that fractionation by partition techniques will be sensitive to the anionic nature of the polymer, but for the common connective tissue glycosaminoglycan, there will be no fractionation according to molecular weight.

The partition of glycosaminoglycan-quaternary ammonium complexes: I. The effect of phase composition

The complexes formed between quaternary ammonium cations and polyanionic glycosaminoglycans can be partitioned between partially miscible aqueous inorganic salt and alcohol phases. Small changes in salt concentration can completely shift the complex from one phase to the other. The effect of the phase composition variables: the type of inorganic salt, the type of quaternary ammonium salt, and the alcohol used, were systematically investigated. The sharp transition from solubility in the upper non-aqueous phase to solubility in the lower, aqueous phase was found to be strongly affected by the type of inorganic salt. This transition occurred at higher salt concentrations when NaCl, KCl, or LiCl were used than when CaCl2 or MgCl2 were used. Differences in behavior among glycosaminoglycans were larger for NaCl than for CaCl2. The complex is stabilized to dissociation by salt by increasing hydrophobicity of the non-aqueous phase. However, aggregation of the complex into an insoluble form is also favored by an increasingly hydrophobic environment. The most consistent partition was observed with 1- and 2-butanol. The partition isotherm of chondroitin 4-sulfate was investigated at constant salt concentration. It was found that the partition coefficient varies with the concentration of chondroitin 4-sulfate, although the magnitude of this effect could be diminished by increasing the quaternary ammonium salt concentration.

Countercurrent chromatography--a new method for the fractionation of glycosaminoglycans.

Abstract A new method is described for continuous flow fractionation of glycosaminoglycans (GAG) by partition in two-phase systems. In the 1-butanol/aqueous NaCl two-phase system containing hexadecylpyridinium chloride fractionation is dependent primarily upon chemical composition, or charge density, of the GAG and is relatively independent of molecular weight. Chondroitin 4-sulfate was fractionated according to degree of sulfation and could be completely resolved from heparin. A heparin sample was shown to contain three discrete components differing with respect to sulfaminohexose and sulfate substitution.

The trimethylsilylation reactions of hexosamines, and gas-chromatographic separation of the derivatives

Abstract Several methods for trimethylsilylation were applied to the 2-amino-2-deoxy-hexoses, and the resulting derivatives were characterized by gas-liquid chromatography as to retention index, response factor, and degree of substitution on the amino group. Conditions were found for replacing none, one, or both of the amino protons with trimethylsilyl groups. The average response-factor for the trimethylsilyl derivatives was found to be 6.27 g of docosane per mole of hexosamine, or 1.13 mg of docosane per mg of hexosamine. One of the methods investigated was applied to the determination of hexosamines in acid hydrolyzates of glycosaminoglycans. The 2-acetamido-2-deoxy derivatives were also studied, but the several derivatives have not yet been identified.

BIOPHYSICAL CHARACTERISTICS OF ANIONIC DENSITY-FRACTIONATED MUCOSAL HEPARINS IN RELATION TO POTENCIES IN ANTICOAGULANT AND THROMBIN-INHIBITION ASSAYS

Abstract The apparent molecular weights determined by high pressure liquid chromatography (HPLC), specific rotations, anticoagulant potencies and thrombin-inhibition potencies of a series of heparin fractions of different anionic density were compared. The fractions were prepared by sequential extraction of the heparin-quaternary ammonium complexes from butanol with aqueous solutions of successively increasing NaCl concentration. Surprisingly, their molecular weights determined by HPLC varied with anionic density, whereas molecular weights by gel chromatography on agarose did not. The specific rotation of the fractions which contained only heparins was constant at 52°, but those fractions which contained dermatan sulfate showed lower rotations. The anticoagulant potencies of the heparin-containing fractions, measured by the activated partial thromboplastin time (APTT) assay in human plasma, were very similar to previous measurements by the United States Pharmacopeia (USP) method in sheep plasma and, when plotted against the square of the anionic density (Z), showed initial linear portions with a sharp decrease in slope for the higher-charged fractions. In contrast the thrombin-inhibition potency measured in an amidolytic assay system using purified antithrombin and thrombin was linear over the entire range of Z2. The ratio of the thrombin inhibition to anticoagulant potency varied systematically from 0.55 for the heparin fraction with the lowest anionic density to 2.0 for the fraction with the highest anionic density.