Studies on the chemical modification of potato (Solanum tuberosum) lectin and its effect on haemagglutinating activity

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Abstract
Modification of potato (S. tuberosum) lectin with acetic anhydride blocked 5.1 amino and 2.7 tyrosyl groups per molecule of lectin and decreased the hemagglutinating activity of the lectin. De-O-acetylation regenerated 2.0 of the tyrosyl groups and resulted in a recovery of activity. Modification with citraconic anhydride or cyclohexane-1,2-dione did not greatly affect activity, although modification of amino and arginyl groups could be demonstrated. Treatment with tetranitromethane nitrated 3.7 tyrosine residues per molecule of lectin with concomitant loss of activity. The presence of 0.1 M-NN''N"-triacetylchitotriose (a potent inhibitor of the lectin) in the reaction medium protected all the tyrosyl residues from nitration and the lectin was fully active. Modification of tryptophyl groups with 2-hydroxy-5-nitrobenzyl bromide and 2,3-dioxoindoline-5-sulfonic acid modified 0.9 and 2.6 residues per molecule of lectin, respectively, with a loss of activity in each case. Reaction of potato lectin with 2,3-dioxoindoline-5-sulfonic acid in the presence of inhibitor protected 2.4 residues of tryptophan from the reagent. Loss of hemagglutination activity was prevented under these conditions. Reaction of carboxy groups, activated with carbodi-imide, with .alpha.-aminobutyric acid methyl ester led to the incorporation of 5.3 residues of the ester per molecule of lectin. Presence of inhibitor in this case, although protecting activity, did not prevent modification of carboxy groups; in fact an increase in the number of modified residues occurred. This effect could be imitated by performing the reaction in 8M-urea. In both cases the number of carboxy groups modified was close to the total number of free carboxy groups as determined by the method of Hoare and Koshland. Guanidination of lysine residues after carboxy-group modification gave less homoarginine than did the unmodified lectin under the same conditions, suggesting the formation of intramolecular cross-links during carbodi-imide activation. Apparently amino, arginyl, methionyl, histidyl and carboxyl groups are not involved in the activity of the lectin and that tyrosyl and tryptophyl groups are very closely involved. These findings are similar to those reported for other proteins that bind N-acetylglucosamine oligomers and also fit the general trend in other lectins.