Materials and Methods The enzyme assay was carried out using 0.05 M Tris-HCl buffer (pH 9.0) to provide optimal alkaline conditions for enzyme activity. The substrate solution contained 1.00 mM p-nitrophenyl phosphate (pNPP), which is hydrolyzed by calf intestinal alkaline phosphatase (AP) to form the yellow p-nitrophenol (pNP) product. Final enzyme concentrations in the reaction mixtures ranged from 0.0286 to 0.229 g/mL, and a blank containing buffer and substrate but no enzyme was used for baseline correction. A 1.0 M NaOH solution was added at the end of the incubation period to stop the reaction and convert p-nitrophenol to its deprotonated yellow form for spectrophotometric measurement. Each reaction was initiated by adding AP to the substrate-buffer mixture and allowed to proceed for 5 minutes at room temperature. Absorbance readings were taken immediately at 410 nm using a spectrophotometer. p-Nitrophenol concentrations were calculated using the Beer-Lambert law (A = LC), where the molar absorptivity () was 16.2 mM cm and the path length (L) was 1.00 cm. Values were corrected for dilution caused by NaOH addition, and reaction velocity (M min) was determined by dividing the corrected p-nitrophenol concentration by the 5-minute reaction time (1). All assays were conducted under fixed