Lectins Of Leaf Pulps Of Aloe Barbadensis Miller

Two lectins were partially purified from leaf pulps of A. vera (A. barbadensis, cultivated in Turkey) and designated as Aloctin I and Aloctin II (Akev and Can, 1999). The lectins agglutinated rabbit erythrocytes but the hemagglutination by Aloctin I was not inhibited by any of the 20 sugars tested. The two lectins did not possess any glyco-sidase activities.

Chemical properties and sugar-binding specificity

Aloctin I and II were a glycoprotein containing 5% and 4.6% sugars, respectively, and the hemagglutinating activity by Aloctin I was inhibited by N-acetyl-D-galactosamine.

Preparation

The gel was removed from A. vera leaves by scraping with a spoon and the remaining pulps were homogenized with PBS. The supernatant (crude leaf pulp extract) of the extract was obtained by centrifugation (45,700Xg) at 2 °C and was fractionated with 50% ammonium sulfate. The precipitate was separated by centrifugation at 2 °C, suspended in PBS and dialysed against the same buffer. The dialysate (50% ammonium sulfate fraction) was applied to hydroxyapatite column chromatography. The separation was performed by stepwise elution with phosphate buffer (pH 7) and two protein peaks showing hemagglutinating activity were eluted with 5 mM and 20 mM; the former was named Aloctin I and the latter Aloctin II.

Hemagglutinating activity

Hemagglutinating activity was examined towards rabbit erythrocytes. In comparison with rabbit erythrocytes, the activities of the leaf pulp lectins were tested on human erythrocytes of blood group types A Rh (+), B Rb (+), O Rb (+) and O Rh (—) as well as rat erythrocytes. Neither of the two lectins agglutinated any of the blood groups of human erythrocytes and the activity toward rat erythrocytes was weaker than that toward rabbit erythrocytes.

Factors influencing hemagglutination. i Sugars

Twenty sugars (D(+)-glucose, D(+)-galactose, D(+)-glucosamine HCl, D(+)-galactosamine HCl, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D(+)-mannose, D(—)-fructose, D(—)-ribose, L(+)-rhamnose, L(+)-arabinose, D(+)-fucose, D(+)-xylose, D(+)-saccharose, D-maltose, D(+)-cellobiose, D(+)-melezitose monohydrate, D(+)-melibiose monohydrate, D(+)-trehalose dihydrate, D(+)-raffinose pentahydrate) were examined for hemagglutination inhibition; none of the sugars tested inhibited the hemagglutinating activity of Aloctin I up to a 500 mM concentration. Aloctin II was weakly inhibited by N-acetyl-D-galactosamine at 250mM concentration.

Factors influencing hemagglutination. ii Metal ions, heat treatment

Of the 10 metal cations (Mg2+, Ca2+, Ba2+, Mn2+, Fe3+, Co2+, Hg2+, Al3+ [sulfate], Al3+ [nitrate], Pb2+) tested, only Al3+ salts activated the hemagglutination of the two lectins. Aloctin I was heat stable up to 60 °C and hemagglutinating activity was not completely lost even when heated at 100 °C for 1 hour.

Glycosidase activity

Glycosidase activity, such as a- and $-galactosidase and a- and $-glucosidase activities, were examined to know whether lectins themselves possess glycosidase activity, since studies with some Leguminosae seeds showed that a-galactosidase activity was exactly copurified with hemagglutinating activity. The two activities, i.e. the glycosidase and hemagglutinating activities, were separated in different protein peaks obtained through hydroxyapatite chromatography, suggesting that Aloctin I and II does not have glycosidase activity and that the consideration that lectins may in general be plant enzymes (Dey etal., 1986) does not apply to these two aloe lectins.

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