Lower inhibition of fu-cose-PAA-biotin binding was achieved at >10 mM concentrations of maltose, mannose, glucose, and fucose with EC50 values of 31.12 ± 3.6, 218.6 ± 41.4, 85.81 ± 4.2, 162.6 ± 28.1 mM, respectively (Fig. 3).
Mannan and fucoidan were the most efficient carbohydrates blocking fucose-PAA-biotin binding to immobilized SPM. Therefore, the binding affinities of immobilized SPM for biotinylated mannan and fucoidan were tested. Polymeric biotinylated conjugates of mannose (mannan-BSA-biotin) and fucose (fucoidan-biotin) showed a high binding affinity to SPM from uncapacitated sperm (Fig. 4).
Both biotinylated oligosaccharides reached their maximum binding (Bmax) at a concentration of approximately 0.25 |xg/ml, with Bmax values for mannan-BSA-biotin and fucoidan-biotin of 2.25 ± 0.07 and 2.96 ± 0.09 mean arbitrary units ± SD, n = 3, respectively. However, fucoidan-biotin presented the strongest binding affinity to immobilized SPM (mean Kd ± SD from three experiments were 1.40 ± 0.37 and 27.88 ± 3.83 |xM, respectively). When the competition in this binding with fucose and mannan was evaluated, fu-cose failed to compete in fucoidan-biotin binding to immobilized SPM, whereas mannan clearly inhibited this interaction (Fig. 5), presenting an inhibitory effect at concentrations higher than 5 |xg/ml (EC50 values for mannan was over 200 mg/ml).
FIG. 4. Binding of fucoidan-biotin and mannan-BSA-biotin (both 0.00.5 |xg/ml) to the SPM (0.125 ^g/well) from uncapacitated sperm. Both fucoidan-biotin (Fdan) and mannan-BSA-biotin (Mann) did bind to the SPM in a concentration-dependent manner. The data indicated in the figure are corrected for nonspecific binding of biotinylated carbohydrate to noncoated wells. Results shown are representative of three independent experiments, each done in triplicate. Mean values ± SD (n = 3) are indicated.