Galactosidase, Beta Assay

Method: Essentally that of Craven et al. (1965). One unit causes the hydrolysis of one micromole of o-nitrophenyl-β-D-galactopyranoside per minute at 25°C and pH 7.5 under the specified conditions.

Reagents

• 0.3 M Sodium phosphate buffer, pH 7.5 with 0.003 M magnesium chloride
• Substrate diluent: 0.01 M Tris⋅acetate pH 7.5 with 0.01 M magnesium chloride
• Enzyme diluent: 0.01 M Tris⋅HCl, pH 7.5 with 0.01 M magnesium chloride, 0.01 M mercaptoethanol and 0.01 M sodium chloride
• 0.10 M Sodium/potassium phosphate buffer, pH 7.0
• 1.0 M Mercaptoethanol
• 0.014 M o-Nitrophenyl-β-D-galactopyranoside (ONPG) in substrate diluent. Prepare fresh daily.

Enzyme

Prepare a one mg/ml stock solution in 0.10 M sodium/potassium phosphate buffer pH 7.0. Immediately prior to use, dilute further to 0.02 - 0.04 ΔA/min. in enzyme diluent. The protein concentration of the chromatographically purified enzyme (Code: BGC) may be determined as follows:

Note: This enzyme is not stable when diluted. All dilutions should be made as quickly as possible and used immediately.

Procedure

Adjust spectrophotometer to 405 nm and 25°C.

Pipette into cuvette as follows:

0.3 M sodium phosphate buffer	1.0 ml
1.0 M mercaptoethanol	0.3 ml
0.014 M ONPG	0.5 ml
Reagent grade water	1.1 ml

Incubate in spectrophotometer at 25°C for 3 - 5 minutes to achieve temperature equilibration and establish blank rate, if any. Add 0.1 ml freshly diluted enzyme and record increase in A₄₀₅ for 4 - 5 minutes. Calculate ΔA₄₀₅/min from initial linear rate.

Calculation