3.1.1. Standard Curve
1. To 1 mL of whole milk add 0.01-0.25 mL standard solution of 1 mM ^-nitrophenol.
2. Made up to a final volume of 4 mL with 0.5 M Na2CO3.
3. Centrifuge twice for 20 min at 3000g.
4. Determine the extinction coefficient at 410 nm and plot the OD against nM of^-nitrophenol.
5. One unit of ^-glucuronidase is defined as nM of ^-nitrophenol released from the substrate per mL of milk per min.
3.1.2. Enzyme Assay Procedure
1. Remove milk fat from the samples by cool centrifugation at 0°C and 10,000g (see Note 1).
2. To 0.4 mL of milk add 0.2 mL of 40 mM pnPG (1.5 g _p-nitrophenol |3-D-glucuronide in 100 mL of distilled water) and 0.4 mL of 1 M acetate buffer, pH 4.0.
3. Incubate the mixture for 4 h at 50°C.
4. Stop the reaction with 4 mL of 0.5 M carbonate buffer, pH 10.0.
5. Centrifuge twice for 20 min at 3000g. Measure the ^-nitrophenol liberated by the substrate in the supernatant against a blank with nonincubated milk (instead of the substrate solution) in a spectrophotometer at 410 nm (see Note 2).
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