A final concentration, 0.2 |xg/ml, of neutravidin-HRP was prepared in washing buffer and 50 ^l/well was added. After 1 h of incubation, free neu-travidin-HRP was removed and the plates were washed five times in washing buffer. Immobilized enzyme activity was quantified by adding 150 |xl/ well tetramethylbenzidine reagent (containing 100 |xg/ml tetramethylben-zidine, 1 mM H2O2 in 0.1 M citric acid buffer, pH 4.0). The reaction was stopped by adding 50 |xl of 2 M H2SO4 per well. Absorbance was measured at 450 nm, using a microtiter plate reader (Benchmark, Bio-Rad Laboratories B.V., Veenendaal, The Netherlands). For each set of the experiments, ELCBA was assayed using three separate SPM isolates.
Divalent Cation Requirement for Fucose-PAA-Biotin Interactions with SPM
Microtiter plate wells were coated with SPM (0.5 |xg protein/well) and blocked as described above. Each well was incubated for 2 h with 50 |xl/ well of fucose-PAA-biotin (final concentration 0.25 |xg/ml) in incubation buffer supplemented with different concentrations of divalent cations (serial concentrations in the range of 0-20 mM of either CaCl2, MnCl2, MgCl2, ZnCl2, or 10 mM EDTA). Binding of fucose-PAA-biotin was detected as described above.