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

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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-β- 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 (Δϵ = 1.2 × 10 3 m −1 cm −1). A similar spectrum with a comparable value of Δϵ was obtained with 4-methylumbelliferyl- N-acetyl-β- d-galactosaminide. Binding of methyl-α- d-galactoside, lactose, and N-acetyllactosamine all produced small but equally intense protein difference spectra with a maximum (Δ ϵ = 2.8 × 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 Δ S∘ 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(β → 4)GlcNAc(β1 → 2)Man, Gal(β1 → 4)GlcNAc(β1 → 6)Man, and Gal(β1 → 4)GlcNAc(β1 → 6)[Gal(β1 → 4)GlcNAc(β1 → 2)]Man. The titrations were performed at two temperatures to determine the thermodynamic parameters. In the series N-acetyl- d-galactosamine, methyl-α- d-galactoside, and lactose, −Δ H∘ 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 Δ S∘ values which became more unfavorable in the above series (−23 to −101 ± 4 J mol −1 K −1); the most negative value of Δ S∘ was observed with N-acetyllactosamine and the three N-acetyllactosamine-containing oligosaccharides.