Amylase, Beta Assay

Method: That of Bernfeld (1955) wherein the rate at which maltose is released from starch is measured by its ability to reduce 3,5-dinitrosalicylic acid. One unit releases one micromole of β-maltose per min at 25°C and pH 4.8 under the specified conditions.

Reagents

• 0.016 M Sodium acetate, pH 4.8
• 2 N Sodium hydroxide
• Dinitrosalicylic acid color reagent. Prepare by dissolving 1.0 gm of 3,5-dinitrosalicylic acid in 50 ml of reagent grade water. Add slowly 30.0 gms sodium potassium tartrate tetrahydrate. Add 20 ml of 2 N NaOH. Dilute to a final volume of 100 ml with reagent grade water. Protect from carbon dioxide and store no longer than 2 weeks.
• 1% Starch: Prepare by dissolving 1.0 gram of soluble starch (Merck) in 100 ml of 0.016 M sodium acetate buffer pH 4.8. Bring to a gentle boil to dissolve. Cool and, if necessary, dilute to 100 ml with reagent grade water. Incubate at 25°C for 4-5 minutes prior to assay.
• Maltose stock solution, 5 micromoles/ml. Prepare by dissolving 180 mg maltose (MW 360.3) in 100 ml reagent grade water. Incubate at 25°C for 4-5 minutes prior to assay.

Enzyme

Dilute to a concentraton of 1-10 micrograms per ml. A minimum of three different concentrations in this range should be run.

Procedure

Adjust spectrophotometer at 540 nm and 25°C.

Using the maltose stock solution prepare a maltose standard curve as follows: In numbered tubes, prepare 10 maltose dilutions ranging from 0.3 to 5 micromoles per ml. Include two blank tubes with reagent grade water only. Into a series of corresponding numbered tubes pipette 1 ml of each dilution of maltose. Add 1 ml of dinitrosalicylic acid color reagent. Incubate in boiling water bath for 5 minutes and cool to room temperature. Add 10 ml reagent grade water to each tube and mix well. Read A₅₄₀. Plot A₅₄₀ versus micromoles maltose.

Enzyme assay: Pipette 0.5 ml of respective enzyme dilutions into a series of numbered test tubes. Include a blank with 0.5 ml reagent grade water. Incubate tubes at 25°C for 3-4 minutes to achieve temperature equilibration. At timed intervals, add 0.5 ml starch solution (at 25°C). Incubate exactly 3 minutes and at timed intervals add 1 ml dinitrosalicylic acid color reagent to each tube. Incubate all tubes in a boiling water bath for 5 minutes. Cool to room temperature and add 10 ml reagent grade water. Mix well and read A₅₄₀ versus blank. Determine micromoles maltose released from standard curve.

Calculation