Supplementary DataOf

A new Phaseolus vulgaris lectin induces selective toxicity of human liver carcinoma Hep G2 cells

Evandro Fei Fang a, Wen Liang Pan a,Jack Ho Wong a, Yau Sang Chan a, Xiu Juan Ye a, Tzi Bun Ng a,*

a School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong

Phone: 852-26098031; Fax: 852-26035123; E-mail:

Fig. 1Tests of thermal and pH stability of BTKL. (a) pH stability of BTKL was measured after incubation at different pH values for 0.5 h at 37 °C. (b) Temperature stability was tested after incubation for 30 min from 0 to 100 °C. Results represent mean±S.D. (n=3)as percent of the value at 30 °C or pH 7. Asterisk, p < 0.05 compared with the value at 30 °C or pH 7.

Fig. 2Dose response of BTKL induced nuclear morphological changes in Hep G2 cells. After treatment with different doses of BTKL including (a) 0 μM (control), (b) 7.5μM, (c) 15μM, (d) 30 µM, (e) 60 µM, and (f) 90μM for 24 h, Hep G2 cells were stained with 1 μg/mL Hoechst 33342 for 10 min and the morphological changes of cells were examined by fluorescence microscopy. Magnified images of the areas indicated by hollow triangles are presented in Figure 3D. Bar,50 μm.

Fig.3Lipopolysaccharide (LPS) induced production of NO (indirectly tested by the concentration of nitrite and nitrate).(A) Male mouse macrophageswere treatedwith LPS (final concentrations from7.8 to 250 μg/mL) or LPS +polymyxin B sulfate (final concentration 10 U/mL) or LPS + dexamethasone (final concentration 10 μM). The value ofeach data point represents mean plus or minus S.D. of three independent experiments.Asterisk, p< 0.05, LPS +polymyxin B sulfate-treated group, or LPS + dexamethasone-treated group compared withLPS-treatedgroupat the same LPS concentration.(B) Dexamethasone at the concentration (10 μM) used in the current in vitro system had no detectable effect on the NO level.

Table 1Summary of purification steps of BTKL from 350 g dried seeds

Purification step
(active fraction) / Protein
(mg) / Specific activity#
(Units/mg) / Recovery of activity (%) / Purification fold*
Aqueous extract / 3865 / 64 / 100 / -
SP-bound fraction / 850 / 256 / 88 / 4.0
Blue-bound fraction / 565 / 323 / 74 / 5.0
Mono S(MS-3) / 156 / 825 / 52 / 12.9
Superdex 75 (SUP-1) / 81 / 1280 / 42 / 20.0

# Hemagglutinating activity towards rabbit erythrocytes was calculated as mentioned above and *purification fold equals value of specific activity of the chromatographic fraction divided by value of specific activity of crude extract.

Table 2 Hemagglutinating activity of BTKL toward erythrocytes of different origins

Species / Strain/Line / Hemagglutinating activity (Titers/mg)
Human
A / Asian / 1280
B / Asian / 1280
AB / Asian / 1280
O / Asian / 2560
Animal
Rabbit / New Zealand White / 1280
Mouse / BALB/c / 2560
Rat / S/D (Sprague Dawley) / 2560

Table 3Effects of sugars and cations on hemagglutinating activity of BTKL

Effects of carbohydrates and cations
Carbohydrates / Minimum inhibitory concentration (mM) / Cations / Minimum inhibitory concentration (μM)
α-D-(+)Melibiose / − / Na2S2O5 / --
α-Lactose / − / KCl / --
α-L-Rhamnose / − / MgSO4 / 625
D-(+)-Galactose / − / CaCl2 / 39
D-(+)-Glucosamine / − / MnSO4 / 312
D-Manitol / − / FeSO4 / 1250
D-(+)-Mannose / − / CuSO4 / 625
D-(+)-Raffinose / − / CuCl2 / 312
D-(+)-Xylose / − / ZnSO4 / 312
D-Galactonic Acid / − / Pb(CH3COO)2 / 1250
L-(+)-Arabinose / − / MgCl2 / 312
Polygalacturonic Acid / 25.0 / FeCl3 / 39

−, inhibitory effect of the carbohydrate on hemagglutinating activity of BTKL (initially 128 units) was undetectable when the sugar was addedup to a final concentration of 200 mM. --, the reduction of hemagglutinating activityof BTKL caused by EDTA (0.01 M) could not be recovered after adding the cation up to a final concentration of 5 mM.