Folin-Ciocalteu phenol reagent (final concentration 0.1 M) was added for 30 min and color formation was measured in a Beckman DU-62 spectrophotometer (Beckman Instruments Nederland B.V., Mijdrecht, The Netherlands) at 750 nm. ii) Alkaline (EC 18.104.22.168) and acid (EC 22.214.171.124) phosphatases were assayed according to Soucek and Vary. iii) 5′-Nucleotidase (EC 126.96.36.199) activity was determined by incubation of samples in a buffer containing 0.1 M glycine, 10 mM MgCl2, and 5 mM 5′-AMP (pH 8.5) for 30 min at 37°C. The reaction was stopped by addition of phosphate reagent, and phosphate was measured. iv) Acrosin (EC 188.8.131.52) activity was determined by its esterolytic activity on N-a-benzoyl-L-arginine-p-nitroanilide.
The formation of the product p-nitroaniline was measured continuously at 410 nm (e410 = 9.9/mM) during the incubation in a buffer containing 0.1 M Tris and 67 mM NaCl (pH 8.0) at 25°C.
Biotinylation of Mannan and Fucoidan
Biotinylated mannan was prepared by biotinylation of a mannan-BSA conjugate, which was previously prepared. One hundred millimoles NHS-biotin (in DMSO) was diluted to a final concentration of 5 mM in the mannan-BSA solution (10 mg/ml in 0.1 M H3BO3, pH 9), and this mixture was incubated at room temperature for 2 h.