Binding of [1-13C]galactose-labeled N-acetyllactosamine to Erythrina cristagalli agglutinin as studied by 13C-NMR

Abstract

The equilibrium binding kinetics of enzymatically prepared N‐acetyllactosamine to the lectin from Erythrina cristagalli have been investigated by 13C‐NMR spectroscopy. Under the experimental conditions used, NMR signals in the spectrum, corresponding to both the free and bound disaccharide species, were observed for the first time. This has permitted the simultaneous determinations of the equilibrium binding constant and the number of binding sites per lectin molecule. At the relatively high lectin concentrations used (0.3–0.87 mM), the association constants determined at 31°C (∼6×103 M−1) are typically lower then those obtained by other methods employing much lower lectin concentrations. Extrapolation of the experimentally observed values to infinite dilution gave a better fit of the data (Ka∼ 1.4×104 M−1) with the binding constant determined by other methods (K∼ 1.1×104 M−1). The sugar residence time on the lectin (∼0.2 s) was determined directly from the signal's line‐width using total line‐shape analysis. Similar NMR experiments may permit an analysis of the interaction of the lectin with glycoproteins and cells labelled with 13C‐enriched galactose residues. Moreover, information on lectin‐galactose interactions at the binding site may be obtained by using galactose labeled at various carbons.

Binding of [1‐13C]galactose‐enriched hen ovalbumin to Erythrina cristagalli agglutinin as studied by 13C‐NMR spectroscopy

“…In principle the apparent number of binding sites per lectin molecule for bulky glycoproteins may be different from that observed for small oligosaccharides. In the case of ECA, the same number of sites is found for the binding of N-acetyllactosamine [16] and for the binding of [l-'3C]Gal-ovalbumin (1.8 vs 2.2, respectively).…”

“…Therefore it could be expected that the value of K. for the binding of N-acetyllactosamine, [l-13C]Gal(P1 -4)GlcNAc, to ECA would be smaller than the K, value determined for the same disaccharide moiety as part of the oligosaccharide side chain of a glycoprotein. Indeed, the K, ( E 6 x lo3 M ) determined by the NMR method for the disaccharide [16] is somewhat smaller than the corresponding value for the glycoprotein, K, z 2 x lo4 M-' as determined in this work. The latter is also in good agreement with the estimated K, w 4 x lo4 M-', at infinite dilution, obtained from the simple competition experiment described in the first part of the previous section.…”

“…In this way average values for n and K, could be obtained by solving all 54 independent sets of linear equations using the 11 experimental points. The dependence of the dissociation equilibrium constant on ECA concentration [16] presents a constraint on the use of Eqn (2) above; however, the lectin concentrations employed in the present study are small so that the contribution of the concentration dependence to the experimental error is estimated (Fig. 3, in [16]) to be about 5%, well within the experimentally determined error.…”

Neoglycoconjugates

“…These reconstructed cell surface glycoconjugates containing only a single specific type of sialyl linkages enabled them to probe specificities of viral binding. In another example of reconstruction of glycoconjugates, Berman et al (Berman et al, 1985;Berman et al, 1986;Berman and Lis, 1987), used a galactosyltransferase to incorporate 13C-labeled Gal onto GlcNAc for binding studies by CMR. Chemical synthesis of such 13C-labeled oligosaccharide chains would be extremely laborious.…”