Expert: Walter Hintz - 1/19/2008

Question
QUESTION: I am doing an experiment to test the effect of different pH levels on the digestion of proteins. The original project that I got the idea from did not have any detailed procedures, so I changed/added on to them and I was hoping if you could see and tell me if the following procedure will work- and if not what I should do to make it work. Thank you very much and please reply as soon as possible! 1.In 100 mL of different pH level BUFFER SOLUTIONS (levels of 2, 4, 7, 10, and 12), add 0.2 grams of powdered albumin. 2.Stir the albumin/buffer solution to form the albumin-buffer solution. 3.Pour 5 mL of the solution in to the test tube. 4.After waiting for 10 minutes, add 2.5 mL of biuret solution. 5.Mix by rotating the test tubes in your palms for 20 seconds. 6.Wait 30 minutes for the color to fully develop. 7.After waiting 30 minutes, test the absorption of light using spectrophotometer to see the range of protein left over in the test tubes. Would putting powered albumin into different pH level buffer solutions actually digest the proteins down? My science teachers have said it SHOULD work, but they are not 100% sure because they have not done/seen the experiment for themselves.

ANSWER: Tell me a bit more about this.  What is your hypothesis? I would suggest that you use an albumin solution rather then dumping in powder.  You seem to be doing things correctly.  I see no reason why this is not succesful.

---------- FOLLOW-UP ----------

QUESTION: My hypothesis is that as the level of the pH level increases, then the rate of the digestion of proteins will increase.

I was instructed by books/internet researches to make albumin solution by diluting albumin with water, but the proteins did not break down. My chemistry teacher told me that I should try mixing the albumin powder directly into the buffer solutions and making it into 1 solution, since diluting the powdered albumin with water then mixing it with buffer solutions will dilute the ph levels because of the water. I did so many different researches online but I cannot find any further information to be helpful. I came across a couple of similar projects but they were from "pay-to-do" sites. If you could suggest anything or catch any errors in doing this experiment it would be a GREAT help. Thank you!

Answer
What you are doing seems logical. What proteolitic enzyme are you using? Since protein digestion takes place mainly in the duodenum I would suspect that digestion would occur at pH levels below 7.0.
You might find some information by investigation of different pancreatic proteases.