Amylase, Alpha Assay
Method: That of (1951) wherein the reducing groups released from starch are measured by the reduction of 3,5-dinitrosalicylic acid. One unit releases from soluble starch one micromole of reducing groups (calculated as maltose) per minute at 25°C and pH 6.9 under the specified conditions.
Dilute to a concentration of 1-10 micrograms/ml. A minimum of three different concentrations in this range should be run.
Adjust spectrophotometer to 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 5.0 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 distilled water to each tube and mix well. Read 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.