The interaction of glycoconjugates with immobilized 5-hydroxytryptamine

Abstract

Interaction between N-acetylneuraminic acid (NeuAc) and 5-hydroxytryptamine (5HT) has been described in biological systems where sialoglycoconjugates participate in highaffinity binding in synaptosomes (Dette & Wesemann, 1978) and platelets (Tamir et al., 1980). The use of immobilized 5HT was described by Sturgeon & Sturgeon (1982), who showed the possibility of fractionating sialic acids and sialoglycoconjugates using this matrix, and we have recently reported a detailed analysis of sialic acid binding to immobilized 5HT (Corfield et al., 1985). These studies have been extended to monitor the specificity of binding to the matrix, in view of reports that 5HT also binds to glycosaminoglycans (Uvnas & Aborg, 1976), to measure the strength of binding and to evaluate the use of this matrix as a preparative and analytical method. Sialic acids, derivatives and sialoglycoconjugates were obtained as reported before (Corfield et al., 1979, 1985). Uronic acids, polyuronic acids and glycosaminoglycans were obtained from the Sigma Chemical Company or prepared. 5HT was immobilized on Sepharose CL-4B as before (Corfield et al., 1985) and contained 3.8pmol of SHT/ml of packed gel. The binding and elution of sialic acids and sialoglycoconjugates was evaluated using columns (2ml) of immobilized 5HT and the compound (0.3-1.5pmol as NeuAc in 1.5 ml of water). Columns were washed with 6ml of water and eluted with a linear gradient of NaCl in water (2 x 50ml). Monosialyl and disialyl compounds were eluted with gradients of NaCl from 0-5 or 0-lOmM, multisialyl compounds with 0-50 or 0-100mM-NaC1 and gangliosides with 0-500 mM-NaC1 followed by a 1M-NaC1 wash. Fractions (2ml) were collected. Uronic acid and compounds containing uronic acid (0.5-2.0pmol in 1.5 ml of water) were loaded onto columns ( lml ) of immobilized SHT, washed with 4ml of water and eluted with two 2 ml aliquots of 5 , 10, 25, 50, 100, 250 and lOOOmM-NaC1 in water. Fractions (2ml) were collected and assayed for sialic acid (Schauer, 1978), uronic acid (Bitter & Muir, 1962) or radioactivity (Corfield et al., 1985). Immobilized 5HT was found to bind uronic acids; glucuronic, galacturonic and mannuronic acids were all bound quantitatively. In addition polygalacturonic acid, polymannuronic acid and polyguluronic acid were quantitatively bound and heparin and chondroitin sulphate behaved in the same way. Only 30-50% of loaded hyaluronic acid (total loaded uronic acid = 1 pmol) was bound. Sialic acid-containing compounds were bound quantitatively when applied at approx. 2mM with respect to sialic acid but less pure samples with an ionic strength above approx. 0.02 mM prevented complete binding of sialoglycoconjugates. The binding of sialic acids, monosialyl compounds and uronic acids was found to be weak. When analysed on gradients of 0-5 mM-NaC1 the following compounds all eluted at 1 mM-NaC1: NeuAc and N-glycolylneuraminic acid (NeuGc), CMP-NeuAc, NeuAca(2-6)-N-acetylgalactosaminitol, NeuAcand NeuCc-a(2-3)-lactose, NeuAca(2-3)galactosylp( 1 -3)-N-acetylgalactosaminitol, monosialyl-lacto-