Purification and characterization of a lectin from Phaseolus vulgaris cv. (Anasazi beans)

Abstract

A lectin has been isolated from seeds of the Phaseolus vulgaris cv. "Anasazi beans" using a procedure that involved affinity chromatography on Affi-gel blue gel, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono S, and FPLC-gel filtration on Superdex 200. The lectin was comprised of two 30-kDa subunits with substantial N-terminal sequence similarity to other Phaseolus lectins. The hemagglutinating activity of the lectin was stable within the pH range of 1-14 and the temperature range of 0-80 degrees C. The lectin potently suppressed proliferation of MCF-7 (breast cancer) cells with an IC(50) of 1.3 microM, and inhibited the activity of HIV-1 reverse transcriptase with an IC(50) of 7.6 microM. The lectin evoked a mitogenic response from murine splenocytes as evidenced by an increase in [3H-methyl]-thymidine incorporation. The lectin had no antifungal activity. It did not stimulate nitric oxide production by murine peritoneal macrophages. Chemical modification results indicated that tryptophan was crucial for the hemagglutinating activity of the lectin.

Figure 1

(a) Fractionation of the crude extract of Anasazi beans on an Affi-gel blue gel column equilibrated with the binding buffer (10 mM Tris-HCl, pH 7.3). The column was washed initially with the binding buffer to remove B1 and then eluted with 1000 mM NaCl in 10 mM Tris-HCl buffer, (pH 7.3) to desorb B2. (b) Superdex 200 column chromatography. Buffer 20 mM Tris-HCl buffer, (pH 7.3), flow rate: 0.5 mL/min, fraction size: 1.0 mL. Only the major peak (SU1) exhibited hemagglutinating activity. mAU = milli-absorbance units.

Figure 2

SDS-PAGE of Anasazi bean lectin. Lane 1: Anasazi bean lectin. Lane 2: molecular mass standards. From top downward, phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa), and α-lactalbumin (14 kDa).

Figure 3

Effect of temperature on hemagglutinating activity of Anasazi bean lectin.

Figure 4

HIV-1 reverse transcriptase inhibitory activity of Anasazi bean lectin (data represent means ±SD, n = 3). IC₅₀ = 7.6 μM. (Data represent means ±SD, n = 3.)

Figure 5

Inhibitory effect of Anasazi bean lectin on proliferation of cancer cell lines. Cell proliferation was determined by MTT assay (Data represent means ±SD, n = 3.)

Figure 6

Mitogenic effect of Anasazi bean lectin and Con A toward mouse splenocytes. (Data represent means ±SD, n = 3.)

Figure 7

Effects of lipopolysaccharide (LPS), Anasazi bean lectin, and dexamethasone on nitric oxide production by mouse peritoneal macrophages. (Data represent means ±SD, n = 3.)

Figure 8

Effect of the modification of tryptophan residues on the hemagglutinating activity of Anasazi bean lectin. (Data represent means ±SD, n = 3.)