Adriana MERIDA, Leonardo VASQUEZ, Margarito MARTINEZ, Teresa AQUINO, Eduardo PEREZ-CAMPOS, and Felix CORDOBA

Biochemistry and Immunology Unit of Technological Institute of Oaxaca, and Departaments of Biochemistry and Experimental Medicine, National University of Mexico School of Medicine, P. O. Box 872, Oaxaca 68000. Oaxaca. Mexico.

Dextran Raffinose Derivative Assay for Affinity Purified Corn Coleoptile Lectin




Biosci. Biotech. Biochem., 60 (12), 2064-2065. 1996.
Received September 21, 1995


Corn coleoptile lectin was isolated using lactose/agarose affinity chromatograpy. The purified lectin, displaying a single band in polyacrylamide gel electrophoresis, was studied by a technique using dextran-raffinose biotinylated derivatives. Raffinose, lactose, lactulose, N-acetyl-D-galactosamine, glucose, and L-fucose were used to measure the inhibition and binding activity of the lectin. The inhibition pattern obtained agreed with previous hemagglutination inhibition data.

Key words: derivative; raffinose; corn; coleoptile; lectin

Due to the capacity of lectins to bind specific carbohydrate structures. purified and commercial lectins are widely used for research on glycoproteins and glycoconjugates, both in solution and on the cell surface. (1-3) Due to the fact that lectins induce in cells various physiological and experimental effects,(4-5) numerous methods, in particular those based on enzyme-linked immunosorbent assay (ELISA) procedures, are used to verify glycoconjugate-lectin interactions. (6-7)

Lectins in corn have been studied by Lepekhin et al. (8) and in corn seeds by Jankovic et al. (9) Also we have described an isolation procedure using red cell stroma to overcome the known instability of the corn coleoptile lectin.(10), Although the hemagglutination assay used before is fairly sensitive and simple, it is rather uncertain due to concomitant large error arising from visual assessment of agglutination of the cells by lectins. (11) Furthermore, the activity of lectins that contain only a single carbohydrate binding site per molecule cannot be detected by this method because of their inability to agglutinate cells.

In this paper we describe an improved technique for the isolation of corn coleoptile lectin by the use of a lactose/agarose affinity column combined with the sugar binding activity of the purified lectin by the dextran derivative biotin assay.(11)

Corn coleoptile (900g, wet weight) was obtained from commercial maize seeds (Asgrow 667 UpJohn, Kalamazoo. U.S.A.) grown in plastic trays in the laboratory in the dark for 6 days. Extracts were obtained using a Waring blender containing 1:1 (w/v) coleoptile and cold extracting buffer. 0.05M KCI. 0.1M sodium citrate, with calcium, magnesium, and manganese chlorides. at 1 mM each. D-Galactose (0.05 M) was added and the pH adjusted to 6.3. The crude extract was filtered, two volumes of chilled acetone were added to the filtrate, and the preparation stored in a refrigerator overnight. The supernatant was poured out and the precipitate collected, dissolved in 118 ml of extracting buffer, centrifuged at 7500 x g in a cold centrifuge. and filtered using a Corning 45 micro m filter unit. A plastic column with a 10 ml reservoir with porous disk (Biorad, U.S.A.) was filled with 1.6 ml of alfa-lactose insolubilized on 6% agarose (Sigma Chem. Co. U.S.A.). The column was placed in a cold cabinet (4 °C) and washed extensively overnight with the same extracting buffer without D-galactose.

The lectin was eluted from the affinity column using extracting buffer with 0.15 M alfa-lactose. Absorbance at 280nm. protein by the Bradford method.(12) and hemagglutination activity were measured as described in reference, (10) using indistinctly washed human A, B, or H(O) erythrocytes with similar results.

Purified corn coleoptile lectin was analyzed in non-denaturing polyacrylamide gel electrophoresis at 7% gel concentration.(13) The gels were scanned using a Helena Junior 24 Densitometer after staining with Brilliant Blue G colloidal Concentrate (Sigma Chemical Co.).

Dextran-raffinose biotinylated derivatives were prepared and used as described by Hatakeyama et al.(11) with minor modifications. Purified corn coleoptile lectin was used at concentrations from 5 to 71 micro g/ml (50 micro l) to coat wells of the polystyrene microtiter plate overnight at room temperature as described by Masso et al (14). Next, the dextran derivative 100 micro l (0.5 to 16 micro g/ml) in TBS-Tween (10mM Tris-HCl, 0.15M NaCl, 0.05% Tween 20), at pH 7.5 with 1 mM each of calcium. magnesium, and manganese chlorides were added. Following incubation for 1 hour at room temperature, the solution was removed. the plates washed several times with TBS-Tween and streptavidin-HRP conjugate from Sigma Chemical Co. (prepared by the method of O'Sullivan et al.(5). One hundred micro l of conjugate (0.6 mg/ml) in TBS-Tween was added, followed by substrate solution (0.006% H2O2 ( Merck ) and O-phenylenediamine 0.6 mg/ml) in citrate phosphate buffer. pH 5.2. The reaction was stopped with 0.1ml of 3.5M H2SO4 and the color measured in an ELISA scanner at 49 nm. Sugar inhibition was obtained by mixing the dextran derivative with increasing amounts of the sugar to be tested, before adding to the lectin adsorbed in the microtiter plate, followed by the same steps as before.

Using this purification procedure, acetone treatment precipitated close to 65% of the hemagglutinating activity in the crude extract. Specific activity increased 34 times and 44.5 mg of active protein were recovered from a total of 2396 mg of protein in the starting material (see Table).

In a second purification step lactose/agarose affinity chromatography was used. The lectin came out as a sharp peak with very high agglutinating activity for human type O red cells and identical high titers for human type B red cells. in spite of the known differences in blood type receptors and the clear specificity of the corn lectin to galactose-containing sugars. (10)

Specific activity in the crude extract was 2.37 x 105 (HAU/mg) hemagglutinating units/mg of protein and 1130 x 105 ( HAU/mg) in the purified lectin. The purification increased about 476 times.

Purity of corn lectin was evaluated by native polyacrylamide electrophoresis. Figure 1 is a Polaroid photo of the gel and a densitometric tracing displaying a sharp single component.

Very pure corn Iectin. obtained as indicated. appeared to be a suitable protein to test the dextran derivative assay described by Hatakeyama et al. in the study of Ricinus communis and soy bean lectins.(11) Figure 2 shows the results obtained using several concentrations of lectin at various concentrations of raffinose derivative from (0.5 to 16 micro g/ml). Although the reaction follows Iectin concentration, the kinetics does not appear linear because of a short lapse initially. Comparable optical densities to the ones presented in (11) are obtained at slightly high corn lectin concentrations. On the other hand. Fig. 3 shows inhibition results with very small amounts of different sugars; as expected, raffinose, lactose, and lactulose came as potent binding sugars. N-acetyl-D-galactosamine a moderate inhibitor and D-glucose and L-fucose with no affect at concentrations even higher than 0.1M. This inhibition behavior closely follows the hemagglutination inhibition data published before, by Lepekin et al. (8) and by Martinez et al.(10).

The technique is reproducible and easy to perform and represents an advance over methods published previously. (4,8)




AcknowIedgments. We thank Dr. Thomas Boone Hallberg for manuscript revision. This study was supported, in part, by research grants from the Universidad Nacional Autonoma de Mexico DGAPA IN 206593, and Consejo Nacional de Ciencia y Tecnologia. Mexico, 2182N-N9304.



Table. Purification of Corn Coleoptile Lectin by Affinity Chromatography on Lactose/Agarosea
Totalactivity (HAU) Total protein (mg) Specific activity (HAUc/mg) Yield (%)
Crude extract 567 x 106 2396 2.4 x 105 100
PP1b 367 x 106 44.5 82.5 x 105 65
Affinity chromatography agarose/lactose 226 x 106 2.0 1130 x105 40
a One kilogram of corn fresh coleoptile was used for lectin purification.
b PP1, precipitation with cold acetone.
c HAU, hemagglutinating activity units.




Fig. 1. (img00003.gif - 9.0 K)
Fig. 1. Native PAGE and Densitometric Tracing of Corn Coleoptile Lectin.



Fig. 2. (img00004.gif - 5.9 K)
Fig. 2. Reaction of dextran-raffinose Conjugate with Pure Corn Coleoptile Lectin.

Lectin was used at 2.5 to 71 micro g/ml concentration in presence of several derivative amounts (numbers in vertical row represent concentration in micro g/ml).




Fig. 3. (img00005.gif - 6.9 K)
Fig. 3. Binding of Dextran-raffinose Derivatives (8 micro g/ml) to Purified Corn Coleoptile Lectin (35 micro g/ml) in the Presence of a Series of Inhibitory Sugars.



Abbreviations: HRP, horseradish peroxidase; TBS, Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis; HAU, hemagglutinating activity units.

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