Purification and Properties of a d‐Galactose/N‐Acetyl‐d‐ galactosamine‐Specific Lectin from Erythrina cristagalli

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Abstract
The lectin from the seeds of Erythrina cristagalli has been isolated in high yield (75 %) and homogeneous form by affinity chromatography on a column of D‐galactose‐derivatised Sepharose. It is a glycoprotein with a molecular weight of 56800±900 and s20.w, = 3.9 S, composed of two subunits (apparent molecular weights of 28000 and 26000 respectively) both of which are glycosylated. The total carbohydrate content is 4.5% and it is comprised of mannose, N‐acetylglucosamine, fucose and xylose in amounts corresponding to 7, 4, 2 and 2 mo1/56800 Da respectively. The amino acid composition of the lectin is characterised by a high content of acidic and hydroxy amino acids, low content of methionine and absence of cysteine. Valine is the only N‐terminal amino acid detected. The lectin is a metalloprotein in that it contains 0.093% Mn and 0.13percnt; Ca (1 mol and 1.9 mo1/56 800 Da respectively), both of which are tightly bound to the protein. E. cristagalli lectin agglutinates untreated human erythrocytes of all blood types, as well as rabbit erythrocytes, at a concentration of 5‐10 μg/ml. It is mitogenic for human peripheral blood T lymphocytes at an optimal concentration of about 100 pμg/ml, but is not mitogenic for mouse thymocytes or splenocytes. d‐GahtOSe and various d‐galactosides inhibit the hemagglutinating activity of the lectin. N‐Acetyllactosarnine is most potent, completely inhibiting four agglutinating units of the lectin at 0.4 mM concentration. Lactose, N‐acetyl‐d‐galactosamine and d‐galactose are 5, 16 and 35 times less active respectively. Lactose specifically perturbs the ultraviolet spectrum of the lectin in the aromatic region. The difference spectrum obtained upon binding of the disaccharide to the lectin shows maxima at 291 nni and 282‐284 nm, indicating a change in the environment of tryptophan residues of the protein upon binding of sugar.