A proteinaceous inhibitor of the digestive enzyme alpha-amylase (1,4-a-D-Glucan glucanohydrolase; EC 3.2.1.1) has been isolated and purified from kidney beans Phaseolus vulgaris. It is a glycoprotein and it specifically inhibits corresponding plant, bacterial and fungal enzymes. Maximum inhibitory activity, against alpha-amylase, occurs at 37° C and pH 5.5. The inhibitor has a molecular weight of 45,000-50,000 (Marshall, JJ and Lauda C.M., J. Biol. Chem., 250, 8030, 1975). Similar alpha-amylase inhibitors also occur in wheat, rye and some other beans (Marshall, J.J., Am. Chem. Soc. Symposium, Series 15, 266, 1975).

One unit amount of inhibitor which, after preincubation for 20 minutes at 25° C, pH 6.9 will reduce the activity of two units of human salivary alpha-amylase by 50% (one unit of alpha-amylase will liberate, from soluble starch, one micromole of maltose per minute at 25° C, pH 6.9).

The reducing groups liberated from starch hydrolysis by salivary alpha-amylase, reduce 3,5-dinitrosalicylic acid resulting in formation of a colored product which can be measured spectrophotometrically at 540 nm. If the alpha-amylase inhibitor is present in the assay system it will inhibit alpha-amylase activity causing a corresponding decrease in the absorbance at 540 nm.

1. 0.02 M Sodium phosphate buffer, pH 6.9 containing 0.006 M sodium chloride.
2. Dinitrosalicylic acid color reagent. Dissolve 1.0 g 3,5-dinitrosalicylic acid in 20 ml 2 M NaOH. Add slowly, 30.0 g sodium potassium tartrate tetrahydrate. Dilute to a final volume of 100 ml with distilled water. Store in a tightly sealed container and protected from CO2. Stable for 2 weeks.
3. 1% Starch. Dissolve 1.0 g soluble starch in 100 ml 0.02 M sodium phosphate buffer, pH 6.9, containing 0.006 M NaCl. Bring to a gentle boil to dissolve. Cool and make volume up to 100 ml, with distilled water, if necessary. Incubate at 25° C for 5 minutes prior to assay.
4. Alpha-amylase/alpha-amylase inhibitor mixture. Prepare this solution fresh. It contains human salivary alpha-amylase (1-2U), alpha-amylase inhibitor (0.5-1U), human serum albumin (1.5 mg), calcium chloride (1.5 mg) in 0.02 solution phosphate buffer, containing 0.006 M NaCl, pH 6.9. Total volume of the mixture is 1.5 ml. This mixture is preincubated for 20 minutes at 25° C prior to assay.

1. Adjust spectrophotometer to 540 nm and 25° C and water bath at 25° C.
2. Pipette 0.5 ml of the preincubated enzyme-inhibitor mixture into a series of numbered test tubes. Include a blank tube containing 0.5 ml distilled water and one containing the preincubated alpha-amylase (control) but without the inhibitor (see Reagent 4).
3. Incubate tubes at 25° C for 5 minutes to achieve temperature equilibration.
4. At timed intervals, add 0.5 ml starch solution (at 25° C) and incubate exactly 3 minutes at 25° C.
5. Exactly 3 minutes following the addition of starch, add 1 ml dinitrosalicylic acid reagent to each tube at timed intervals.
6. Place all tubes in a boiling water bath for 5 min.
7. Cool to room temp and add 10 ml distilled water to each tube. Mix well.
8. Read absorbance at 540 nm after zeroing the spectrophotometer with the blank.
9. Determine micromoles of maltose liberated from a maltose standard curve.