Inhibitory Actions of Pesticides on Alpha-Amylase

[atreyad]

Hi,

My project is comparing different rates of inhibition of pesticides on the digestive enzyme alpha-amylase. I have been testing 7 different insecticides, 3 of which are organically based, and the other 4 are synthesized. I have been visually testing this by using a starch indicator I2/KI, a potassium and iodine solution, and I have been timing the reaction of the enzyme+substrate solution till it becomes clear. I am also intrigued about other digestive enzymes such as lipase, and either trypsin or pepsin; however I do not know of a method to visually test these enzymes if you know of a way to do this testing then it would be very useful.

My second question is how I can test this with a spectrophotometer. I have read many papers on Michaelis Menten kinetics however I am still having some trouble understanding how to actually graph velocity of the reaction. Also how should I go testing the reaction of the enzyme+substrate in the spectrophotometer, I am currently using a spectronic-20 and I am having some trouble measuring the absorbency over time and I think a step by step instruction on how to actually perform the experiment in the spectrophotometer would be very helpful. Please tell me if I have left any specific details out of this very short explanation, thank you very much though!

[amyC]

Hi - Is this a project (and research) you are completing for science fair? I ask because I wasn't sure about your due date, which you have listed as: 14/25/08.

Amy
Science Buddies

[michellebayefsky]

Dear atreyad,

Here is an experiment that uses color change to indicate lipase reactions:
http://www.biotopics.co.uk/nutrition/lipase.html

This experiment uses univeral indicator paper to measure pepsin reactions:
http://www.biotopics.co.uk/nutrition/pepsin.html

The following link explains how to use a spectrophotometer step by step.
http://biology.clc.uc.edu/Fankhauser/La ... ometer.htm

Hope this helps!