Procedure
1.Pipette 4.0 mL of substrate solution (A) into a test tube (32 φ× 200 mm) and equilibrate at 40 ℃ for approximately 5minutes.
Concentration in assay mixture |
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Acetate buffer | 42 mM |
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Starch | 0.8 % |
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2.Add 1.0 mL of the enzyme solution* and mix.
3.After exactly 15 minutes at 40 ℃, add 2.0 mL of alkaline solution (B) to stop the reaction.
At the same time, prepare the blank by first mixing the substrate solution with 2.0 mL of alkaline solution after incubation for 15 min at 40 ℃, followed by addition of the enzyme solution.
4.Add 2.0 mL of CuSO4 solution (C) and, after covering the test tube with a marble (40 mmφ) to prevent evaporation, place the test tube in a boiling water bath.
5.After 20 minutes, remove the test tube from the boiling water bath and cool to room temperature under running water.
6.Add 2.0 mL each of KI solution (D) and H2SO4 solution (E) in this order.
7.Shake the test tube and determine the amount of residual Cu2+ by titration with Na2S2O3 solution (F).
8.Record the titers (mL) of the test (Δt) and the blank (Δb), and calculate the titration difference in mL (Δ sample: Δb−Δt).
*Dissolve the enzyme preparation in ice-cold distilled water and dilute to 0.4−1.5 U/mL with enzyme diluent (G), immediately before assay.
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