*4.4. Enzyme and Protein Essays*

To determine the activity, 0.02 mL of an enzyme solution were mixed with 0.38 mL of the pNP-α-Gal solution (1 mg/mL in 0.05 M sodium phosphate buffer, pH 7.0). The reaction mixture was incubated at 20 ◦C for 10 min. The reaction was stopped by addition of 0.6 mL of 1 M Na2CO3. One unit of activity (U) was determined as the amount of enzyme that releases 1 μmol of pNP per 1 min at 20 ◦C in 0.05 M sodium phosphate buffer at pH 7.0. The amount of released pNP was determined spectrophotometrically (ε<sup>400</sup> = 18300 M−<sup>1</sup> cm−1). The specific activity was calculated as U/mg of protein. Protein concentration was determined by the Bradford method and calibrated with BSA as a standard [58].
