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 (Δ? = 1.2 × 10
3m
?1 cm
?1). A similar spectrum with a comparable value of Δ? 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
? = 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.
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