Enzyme Activity: Pancreatic Amylase

This exercise demonstrates quantitatively the substrate and pH specificity of pancreatic amylase. Hydrolysis of polysaccharide substrate generates new reducing ends which are detected using DNSA reagent and a spectrophotometer. Three glucose-containing polysaccharides are tested as substrates: starch, cellulose, and dextran.

In the first part of the exercise, students determine amylase's substrate specificity. The complete reaction mixture contains one drop of pH 8 buffer (1 M triethanolamine titrated to pH 8 with HCl), six drops of substrate, and one drop of amylase solution. Substrates are 1% soluble starch and 1% dextran (any molecular weight is fine). Cellulose is added as a small piece of cotton with six drops of water. The amylase solution contains 50-70 mg amylase (porcine pancreas, Sigma #A3176) in 100 mL of water.

Eight tests are performed (I recommend the students use 18 x 100-mm test tubes) as follows:

1. Buffer control: 7 drops of water, 1 drop of pH 8 buffer
2. Amylase control: 6 drops of water, 1 drop of pH 8 buffer, 1 drop of amylase
3. Starch control: 1 drop of water, 1 drop of pH 8 buffer, 6 drops of starch
4. Starch + amylase: 1 drop of pH 8 buffer, 6 drops of starch, 1 drop of amylase
5. Dextran control: 1 drop of water, 1 drop of pH 8 buffer, 6 drops of dextran
6. Dextran + amylase: 1 drop of pH 8 buffer, 6 drops of dextran, 1 drop of amylase
7. Cellulose control: a small piece of cotton, 7 drops of water, 1 drop of pH 8 buffer
8. Cellulose + amylase: a small piece of cotton, 6 drops of water, 1 drop of pH 8 buffer, 1 drop of amylase

After adding the amylase, tubes are left standing at room temperature for 10 minutes. Then students add eight drops of DNSA reagent, heat in a boiling water bath for ten minutes, and dilute with 4 mL of water. Absorbance is determined at 540 nm with a Spectronic 20. (The results are evident, however, even without the instrument.)

In the second part of the exercise, students determine the pH optimum of amylase. Here, reactions contain 1 drop of various buffers, 6 drops of 1% starch, and 1 drop of amylase. Incubation and workup are as described above for substrate specificity. Buffers are prepared at concentrations of 1 M as follows:

1. pH 2: equimolar sodium sulfate and sodium bisulfate
2. pH 4: acetic acid adjusted to pH 4 with NaOH
3. pH 6: succinic acid adjusted to pH 6 with NaOH
4. pH 8: (triethanolamine as above)
5. pH 10: sodium bicarbonate adjusted to pH 10 with NaOH
6. pH 12: potassium phosphate (dibasic) adjusted to pH 12 with KOH

In the first part of this exercise, the only tube with significant color contains both starch and amylase. Cellulose contains only beta 1-4 linkages and dextran contains predominantly alpha 1-6 linkages. The second part of the exercise shows amylase to be most active at pH 6 and 8.

After tabulating their absorbances, students are asked (1) what kinds of glycosidic bonds are present in starch (both alpha 1-4 and 1-6), (2) which kind of bond is hydrolyzed by amylase (and how they know from their results), (3) why the "starch only" tube is such an important control in the first part, (4) to graph their pH data, and (5) to indicate whether pancreatic amylase would be very active in the stomach.