Binding of simple carbohydrates and some N-acetyllactosamine-containing oligosaccharides to Erythrina cristagalli agglutinin as followed with a fluorescent indicator ligand.

Erythrina cristagalli agglutinin, a dimeric lectin [J.L. Iglesias, et al. (1982) Eur. J. Biochem. 123, 247-252] was shown by equilibrium dialysis to be bivalent for 4-methylumbelliferyl-beta-D-galactoside. Upon binding to the lectin, this ligand showed a difference absorption spectrum with two maxima (at 322 and 336 nm) of equal intensity (delta epsilon = 1.2 X 10(3) M-1 cm-1). A similar spectrum with a comparable value of delta epsilon was obtained with 4-methylumbelliferyl-N-acetyl-beta-D-galactosaminide. Binding of methyl-alpha-D-galactoside, lactose, and N-acetyllactosamine all produced small but equally intense protein difference spectra with a maximum (delta epsilon = 2.8 X 10(2) M-1 cm-1) at 291.6 nm. Upon binding of N-dansyl-D-galactosamine to the lectin, there was a fivefold increase in fluorescence intensity of this ligand. The association constant for N-dansyl-D-galactosamine was caused by a very favorable delta S degree of the dansyl group without affecting the strictly carbohydrate-specific character of binding. N-Dansyl-D-galactosamine was employed as a fluorescent indicator ligand in substitution titrations. This involved the use of simple carbohydrates, N-acetyllactosamine, and oligosaccharides which occur in the carbohydrate units of N-glycoproteins; the latter were Gal(beta 1----4)GlcNAc(beta 1----2)Man, Gal(beta 1----4)GlcNAc(beta 1----6)Man, and Gal(beta 1----4)GlcNAc(beta 1----6)[Gal(beta 1----4)GlcNAc(beta 1----2)]Man. The titrations were performed at two temperatures to determine the thermodynamic parameters. In the series N-acetyl-D-galactosamine, methyl-alpha-D-galactoside, and lactose, -delta H degrees increased from 24 to 41 kJ mol-1; it increased further for N-acetyllactosamine and then remained unchanged for the N-acetyllactosamine-containing oligosaccharides (55 +/- 1 kJ mol-1. This indicated that the site specifically accommodated the disaccharide structure with an important contribution of the 2-acetamido group in the penultimate sugar. Beyond this, no additional contacts seemed to be formed. This conclusion also followed from considerations of delta S degrees values which became more unfavorable in the above series (-23 to -101 +/- 4 J mol-1 K-1); the most negative value of delta S degrees was observed with N-acetyllactosamine and the three N-acetyllactosamine-containing oligosaccharides.