Maximal binding (Bmax) was 4.71 ± 0.48 and 0.40 ± 0.03 (mean arbitrary units ± SD, n = 3; P < 0.001) for fucose-PAA-biotin and LeA-PAA-biotin, respectively. SPM proteins had approximately two times lower affinity for fucose-PAA-biotin when compared with LeA-PAA-biotin. Half maximal binding (Kd values) was obtained at 2.13 ± 0.35 |xg/ml and 1.16 ± 0.15 |xg/ml (mean values ± SD, n = 3; P < 0.01) for fucose-PAA-biotin and LeA-PAA-biotin, respectively (Fig. 1). From the result of this experiment, we decided to use 0.5 |xg SPM per well and only to use fucose-PAA-biotin for quantification of carbohydrate binding because SPM had a higher binding capacity for this conjugate than LeA-PAA-biotin.
The dependency of binding on divalent cations was tested using a range of 0-20 mM of either Ca2+ Mn2+, Mg2+, Zn2+, or EDTA. Binding of fucose-PAA-biotin to the SPM was dependent on divalent cations and present in all cation buffers tested. Binding was blocked completely in the absence of divalent cations (i.e., in the presence of 10 mM EDTA; P < 0.0001, n = 3; Fig. 2) and decreased 20% in the presence of 5 mM Zn2+ (P < 0.01, n = 3; Fig. 2), while the other divalent cations did not affect fucose-PAA-biotin binding when compared with Ca2+ (P > 0.05, n = 3; Fig. 2).
FIG. 1. Binding of fucose-PAA-biotin and LewisA-PAA-biotin (both 0.02.0 |xg/ml) to the SPM (0.125 ^g/well) from uncapacitated sperm cells. Both fucose-PAA-biotin (Fuc) and LewisA-PAA-biotin (LeA) bind to the SPM in a concentration-dependent manner. The data indicated in the figure is corrected for nonspecific binding of the biotinylated carbohydrate to noncoated wells. Results shown are representative of three independent experiments, each done in triplicate. Mean values ± SD (n = 3) are indicated.
FIG. 2. Binding of fucose-PAA-biotin to SPM in the presence of different divalent cations and EDTA. Microtiter plate wells were coated with SPM (0.5 |j,g/well) and incubated with fucose-PAA-biotin (0.25 |xg/ml) in the presence of 5 mM CaCl2 (Ca), 5 mM MgCl2 (Mg), 5 mM MnCl2 (Mn), 5 mM ZnCl2 (Zn), or 10 mM EDTA. Results shown are representative of three independent experiments, each done in triplicate. Mean values ± SD (n = 3) are indicated. **, Significant reduction of binding in the presence of Zn2+, P < 0.01; and EDTA, P < 0.001).