Lucjan J.J. Hronowski

Quantitation and interaction of glycosaminoglycans with Alcian blue in dimethyl sulfoxide solutions

Abstract An improved method is described for the quantitation of glycosaminoglycans separatedon cellulose acetate, stained with Alcian blue, and dissolved in a dimethyl sulfoxide solution. Standard curves are shown for all eight glycosaminoglycans. It is shown that absorption at the Alcian blue orthochromatic Emax is depressed under conditions which favor formation of dye-glycosaminoglycan complexes. The interaction between Alcian blue and the eight glycosaminoglycans was studied in dimethyl sulfoxide solutions of varying composition. It was shown that the extent of complex formation depends both on the glycosaminoglycan and the composition of the dimethyl sulfoxide solution. A dimethyl sulfoxide solution which contains 0.094 m H2SO4 is described which maximizes dye-glycosaminoglycan dissociation and thus the absorbance. Also, an improved staining method is described which improves dye uptake by the glycosaminoglycans and consequently increases the sensitivity of glycosaminoglycan quantitation.

Characterization of glycosaminoglycan-alcian blue complexes by elution from cellulose acetate utilizing different MgCl2 concentrations

Abstract Glycosaminoglycans subjected to electrophoresis on cellulose acetate are stained by Alcian blue in the presence of different MgCl 2 concentrations. The procedure described solubilizes and elutes off the cellulose acetate strip glycosaminoglycans with critical electrolyte concentrations lower than the concentration of MgCl 2 , leaving stained on the strip glycosaminoglycans with higher critical electrolyte concentrations. This technique is particularly useful for sulfated glycosaminoglycans that are incompletely separated by electrophoresis and is applicable to the partial characterization of glycosaminoglycans down to a 0.1-μg range allowing practical quantitative studies of radioactively labeled glycosaminoglycans produced by low-density cell cultures. The staining technique is examined with respect to the specificity of the glycosaminoglycans eluted, and the nature of the interactions in the staining process is discussed. In addition, a simplified procedure is described for the characterization of glycosaminoglycans using testicular hyaluronidase (EC 3.2.1.35) and chondroitinase ABC (EC 4.2.2.4), which can be used in conjunction with the salt elution technique.

Synthesis and characterization of 1-O-β-lactosyl-(R,S)-glycerols and 1,3-di-O-β-lactosylglycerol☆

Abstract The synthesis of 1- O -β-lactosyl-( R,S )-glycerols was achieved by three methods: ( a ) in 25% yield by the trimethylsilyl trifluoromethanesulfonate-promoted reaction of octa- O -acetyl-β-lactose ( 11 ) with ∼0.5 mol-equiv. of 2- O -benzylglycerol ( 4 ), ( b ) in ∼34% yield by the coupling of 4 with an equimolar amount of hepta- O -acetyl-α-lactosyl bromide ( 12 ) in the presence of mercury(II) cyanide, and ( c ) in ∼50% yield by the coupling of equimolar amounts of 12 and 1,2-di- O -benzyl-( R,S )-glycerols in the presence of mercury(II) cyanide, followed in each case by the removal of the blocking groups. 1,3-Di- O -β-lactosylglycerols were prepared in 21% yield by the coupling of 11 with ∼0.5 mol-equivalent of 4 by method ( a ), and in 38% yield by the coupling of 12 with ∼0.5 mol-equiv. of 4 by method ( b ), followed by the removal of the blocking groups. The configuration of the glycosidic linkage between the lactose units and the glycerol residue was established by high-resolution, two-dimensional 1 H-n.m.r. spectroscopy.

Detection and quantitation of proteoglycans extracted from cell culture medium and cultured cartilage slices

Detection and quantitation of extracted proteoglycans, by staining with the dye Alcian blue on cellulose acetate followed by dissolution of the stained cellulose acetate strips in dimethyl sulfoxide containing 0.5% (v/v) sulfuric acid for absorbance measurement, is described. It is shown that, in the present system, the dye uptake by the proteoglycan is dependent only on the glycosaminoglycan content of the proteoglycan. The method is applied to the quantitation and characterization of proteoglycans and glycosaminoglycans, which have been extracted from radiolabeled bovine ankle cartilage and from mononuclear cell supernatant and which have been separated by DEAE-Sephacel column chromatography. The high sensitivity of the method allows detection of proteoglycans in 25-microliters samples of solutions containing as little as 1 microgram of glycosaminoglycan per milliliter of solution.

Synthesis and characterization of 2-O-β-lactosylglycerol, 1,2-di-O-β-lactosyl-(R,S)-glycerols, and 1,2,3-tri-O-β-lactosylglycerol

Abstract The reaction of 2,3,6,2′,3′,4′,6′-hepta- O -acetyl-α-lactosyl bromide ( 5 ) and 1,3-di- O -benzylglycerol in the presence of mercury(II) cyanide in benzene-nitromethane afforded 1,3-di- O -benzyl-2- O -(2,3,6,2′,3′,4′, 6′-hepta- O -acetyl-β-lactosyl)glycerol (70%), which was converted into 2- O -β-lactosylglycerol. 1,2-Di- O -β-lactosyl-( R,S )- glycerols were obtained by way of the coupling of 5 to either 1- O -benzyl-( R,S )-glycerol or 1- O -benzyl-2- O -(2,3,6,2′,3′,4′,6′-hepta- O -acetyl-β-lactosyl)-( R,S )-glycerols. The most efficient route to 1,2, 3-tri- O -β-lactosylglycerol ( 17 ) involved treatment of 2- O -(2,3,6,2′,3′,4′,6′-hepta- O -acetyl-β-lactosyl)glycerol with 3 mol. equiv. of 5 followed by removal of the blocking groups, to give 17 (47%).

Synthesis and characterization of 1-O-α-lactosyl-(R,S)-glycerols and 1-O-α-lactosyl-3-O-β-lactosyl-(R,S)-glycerols

Abstract Coupling of 2,3,6,2′,3′,4′,6′-hepta- O -benzyl-α-lactosyl bromide with an equimolar amount of 1- O -acetyl-2- O -benzyl-( R,S )-glycerols in the presence of tetraethylammonium bromide and 4A molecular sieves in 1,2-dichloroethane afforded 3- O -acetyl-2- O -benzyl-1- O -(2,3,6,2′,3′,4′,6′-hepta- O -benzyl-α-lactosyl)-( R,S )-glycerols in 20.4% yield, which were deprotected to give 1- O -α-lactosyl-( R,S )-glycerols. 2- O -Benzyl-3- O -(2,3,6,2′,3′,4′,6′-hepta- O -acetyl-β-lactosyl)-1- O -(2,3,6,2′,3′,4′,6′-hepta- O -benzyl-α-lactosyl) -( R,S )-glycerols were obtained in 61% yield by the reaction of hepta- O -acetyl-α-lactosyl bromide and 2- O -benzyl-1- O -(2,3,6,2′,3′,4′,6′-hepta- O -benzyl-α- lactosyl)-( R,S )-glycerols in the presence of mercury(II) cyanide in benzene-nitromethane; deprotection afforded 1- O -α-lactosyl-3- O -β-lactosyl-( R,S )-glycerols in an overall yield of 24%. The two target materials are useful in the assessment of the binding properties of d -galactosyl-terminated glyceryl glycosides to the asialoglycoprotein receptor of normal rabbit and human hepatocytes.

Binding of D-galactose-terminated ligands to rabbit asialoglycoprotein receptor.

The binding affinities of a series of D-galactose-terminated glycerol glycosides and oligosaccharides for the asialoglycoprotein receptor isolated from rabbit liver were determined in vitro using a radioreceptor-inhibition assay with 125I-asialoorosomucoid. The relative affinities of the synthetic ligands increased with the number of exposed D-galactose termini. Of the compounds examined, 1,2,3-tri-O-beta-lactosylglycerol associated with the greatest affinity (estimated Kd = 7.97 x 10(-5) M). Examination of the affinities of the synthetic series indicated that both the number and propinquity of the D-galactose termini influenced the strength of the binding interactions.

SYNTHESIS OF NEW CARBOCYCLIC ANALOGUES OF 3'-AZIDO- AND 3'-AMINO-2',3'-DIDEOXYNUCLEOSIDES

A route is described for the synthesis of cyclopentane analogues of 1-(3′-azido- and 3′-amino-2′,3′-dideoxy-β-erythro-pentofuranosyl)-5-iodouracil and -(E)-5-(2-bromovinyl)uracil, compounds of interest as potential antiviral and/or antitumour agents.

Nonspecific interaction of proteoglycans with chromatography media and surfaces: Effect of this interaction on the isolation efficiencies

Nonspecific adsorption of proteoglycans to chromatography media and surfaces is demonstrated. This adsorption is highly dependent on the nature of the chromatography media and the precise buffer conditions. For a given buffer the amount of adsorption decreases as the pH of the buffer is increased. It is also highly dependent on buffer concentration and increases as the buffer concentration is increased. The effect of salts such as LiCl, NaCl, KCl, and MgCl2 was generally small and complex so that the presence of the salt both increased and decreased the amount of adsorption depending on the buffer conditions. In contrast, the effect due to the presence of guanidine hydrochloride (Gdn-HCl) was relatively large and complex. At low Gdn-HCl concentrations there generally was a large increase in the amount of adsorption, reaching a maximum at approximately 0.5 M Gdn-HCl and decreasing with further increases in Gdn-HCl concentration. Detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) and sodium dodecylsulfate generally reduced the amount of nonspecific adsorption, although in the presence of both the detergent and Gdn-HCl, the effect due to Gdn-HCl predominated. In commonly used buffers such as 0.5 M sodium acetate (NaOAc), pH 7.0 (buffer F), and 4 M Gdn-HCl in 0.05 M NaOAc, pH 5.8 (buffer D), adsorption to surfaces and chromatography media such as Sepharose CL-2B, cellulose, and controlled pore glass (CPG) is highly significant and it is particularly large for cellulose and CPG.(ABSTRACT TRUNCATED AT 250 WORDS)

Non-specific interaction of proteoglycans with surfaces and matrices

Evidence is presented that reversible non-specific adsorption of proteoglycans (PGs) to surfaces and matrices is an inherent property of the PGs. This adsorption is dependent on the intact PG structure as the glycosaminoglycans (GAGs), which are isolated after papain digestion of the PG show no such non-specific adsorption. The interaction of the PG with surfaces and matrices is also highly dependent on the internal milieu and can be both inhibited and enhanced by such factors as the ionic composition and concentration, pH, detergents and chaotropic reagents such as guanidine hydrochloride (Gdn-HC1). It is suggested that this inherent stickiness of the PGs allows them to function like a reversible fluid adhesant in the connective tissues. This weak binding force thus not only aids in maintaining the integrity of the connective tissues, but its reversible nature may provide for easy movement of other materials through the connective tissue matrix.