Sven Gardell

Determination of glycosaminoglycans (mucopolysaccharides) from tissues on the microgram scale

Microgram quantities of glycosaminoglycans can be quantitatively liberated from tissues, fractionated and determined by the following procedure: (1) Digestion of the tissue sample with papain (EC 3.4.4.10). (2) Precipitation of the glycosaminoglycans as their cetylpyridinium complexes on an inert support of powdered cellulose packed in a column. (3) Fractional elution of the complexes from the column with salt solutions of increasing concentration. (4) Estimation of the amount of glycosaminoglycans in each fraction after hydrolysis followed by determination of the hexosamines by a modified Elson and Morgan method. Experimental data are presented to validate the various steps of the procedure. The results were found to be reproducible and accurate for fractions down to 2–5 μg of hexosamine. Small differences in the solubility properties of the glycosaminoglycan-cetylpyridinium complexes could be detected and visualized by the construction of “solubility profiles”. Application of the procedure to histological sections of the intervertebral disc and the nasal septum cartilage revealed differences in the amount and properties of glycosaminoglycans in tissue structures 100–200 μ apart. The method should be useful to study the distribution of glycosaminoglycans at the tissue level.

The precipitation of polyanions by long-chain aliphatic ammonium compounds. IV. Elution in salt solutions of mucopolysaccharide-quaternary ammonium complexes adsorbed on a support.

Abstract A technique is described by which detailed information of the composition of an acidic polysaccharide mixture can be obtained. The polysaccharide-cetylpyridinium complex is precipitated on an inert support packed in a column, and is then eluted from the column in salt solutions of increasing concentration. The technique has been applied to the resolution of mixtures of known polysaccharides as well as to less well defined polysaccharide mixtures obtained from various tissues. A micromodification of the method allows as little as 25 μg of each polysaccharide component to be separated and determined.

Extraction and purification of proteoglycans from various types of connective tissue

Abstract A new procedure for the isolation of proteoglycans has been described. Tissues are extracted with 4 M guanidinium chloride. the extracting solven is then exchanged for 7 M urea and the extract is chromatographed on a DEAE-cellulose column previously equilibrated with 7 M urea. Non-proteoglycan proteins were eluted with urea in weak salt solutions. Subsequently proteoglycans were eluted with strong salt solutions. By the procedure proteoglycans from tissues containing only small amounts of proteoglycans can be obtained virtually free from collagen in a form suitable for further fractionation.

Isolation and characterization of glycosaminoglycans from human leukocytes and platelets.

Abstract Glycosaminoglycans have been isolated from human leukocytes and platelets. The polysaccharides were liberated from the disintegrated and fat-extracted cells by digestion with papain. Deoxyribonuclease and ribonuclease were used to remove nucleic acids and the polysaccharides were precipitated with cetylpyridinium chloride. Only about 20–25% of the total hexosamine content of leukocytes and platelets was associated with glycosaminoglycans and the rest was considered to be derived from glycoproteins. The largest part of the glycosaminoglycans obtained from both leukocytes and platelets was identical with chondroitin 4-sulfate but a minor component was probably hyaluronic acid. The biological function in the cell of these substances is still unknown.

An ion-exchange column chromatographic method for the separation and quantitative analysis of neutral monosaccharides☆

Abstract A method for the separation and quantitation of micromole quantities of many of the naturally occurring neutral monosaccharides has been described. This method is based on the ion-exchange chromatography of the sugar-borate complexes on a strong anion-exchange resin. The utilization of a boric acid/glycerol buffer has allowed the chromatographic separation to be performed at pH 6.8 and at an elevated temperature. This system possesses a high degree of resolution and permits neutral monosaccharides to be quantitated with a precision of ±5% or better in the case of some monosaccharides.

SEPARATION OF THE GLYCOSAMINOGLYCANS (MUCOPOLYSACCHARIDES) FROM AORTA BY A COLUMN PROCEDURE USING QUATERNARY AMMONIUM COMPOUNDS.

Summary The glycosaminoglycans (mucopolysaccharides) from human aortas have been prepared and analysed by: (1) Digestion of the tissue with papain. (2) Precipitation of the polysaccharides with cetylpyridinium ions. (3) Fractionation on a cellulose column of the cetylpyridinium complexes of the glycosaminoglycans with MgCl 2 of different concentration and at different pH. (4) Characterization of the fractions by chemical analysis, susceptibility to testicular hyaluronidase and infrared spectroscopy.

Biosynthesis of glycosaminoglycans (mucopolysaccharides) in human leukocytes

Abstract 1. 1. The capacity of human peripheral leukocytes (granulocytes) to synthesize glycosaminoglycans has been studied. 2. 2. Leukocyte suspensions were incubated with [35S]sulfate or [1−14C]glucosamine. The glycosaminoglycans were liberated by papain digestion and fractionated on cellulose columns by cetylpyridinium chloride and salt solutions. The radioactivity of the isolated glycosaminoglycans was determined. 3. 3. It was found that both epimerization of glucosamine to galactosamine and subsequent formation of the chondroitin sulfate chain, as well as sulfation of the latter, occurs. 4. 4. The results also indicate that several pools of glycosaminoglycans with different metabolic activities are present in leukocytes.

Chromatography of glycosaminoglycans on ECTEOLA-cellulose columns.

Abstract Artificial and naturally occurring mixtures of glycosaminoglycans were fractionated on ECTEOLA-cellulose columns. In general galactosaminoglycans could be eluted from the columns with lower concentrations of ammonium formate in ammonia than were needed to elute keratan sulfate. A keratan sulfate that contained, in addition to glucosamine, small amounts of galactosamine but no hexuronic acid, was isolated from nucleus pulposus and nasal septum cartilage.

Analysis of aortic glycosaminoglycans from various animal species by CPC-Cellulose column procedures

Summary Aortic tissue from several species, including some common laboratory animals, has been investigated with regard to types of glycosaminoglycans present and with regard to their separation by the CPC (cetylpyridinium chloride) procedure of Antonopoulos et al . The polysaccharides were liberated from the tissue by papain digestion and precipitated with CPC. The glycosaminoglycan mixture was separated into components by precipitation on a CPC saturated cellulose column, followed by fractional solubi-lisation of the CP-polysaccharide complexes by salt solutions of increasing concentrations. The fractions isolated were analysed by chemical and enzymatic procedures and were also refractionated by an analogous CPC micro procedure. Hyaluronic acid, heparan sulphate, chondroitin sulphates and dermatan sulphate were identified in all materials investigated. The galactosaminoglycans could not be successfully separated: the isometric chondroitin sulphates were quite inseparable, while a purified dermatan sulphate fraction could be obtained only at the expense of considerable loss of material. The results suggest a pronounced polydispersity of the aortic galactosaminoglycans with regard to chain length and/or degree of sulphation. It is stressed that glycosaminoglycan heterogeneity must be taken into account when attempts are made to identify these substances by means of the solubility characteristics of their CP complexes.

Purification of the mitosis-stimulating factor from Phaseolus vulgaris

Abstract The mitosis-stimulating factor from the phytohaemagglutinin of red kidney beans ( Phaseolus vulgaris ) has been purified by: (1) extraction of the beans with phosphate buffer; (2) removal of inert proteins by heat coagulation at 80° for 10 min; (3) ethanol fractionation at 2° at pH 6.8; (4) column chromatography on calcium phosphate. The material finally obtained was of protein or polypeptide nature and contained 6% carbohydrate. Maximal number of mitosis was induced in cultures of growing lymphocytes with only 0.2 μg/ml culture medium. The haemagglutinating property was still present but could be removed by adsorption with red blood cells without any detectable loss of the mitosis-stimulating activity.