an enzyme lab

**Math Is Hard** · Mar7-06, 01:26 AM

We had a lab tonight involving enzymes. I am concerned about two of the results we got.

Test tube #1 contained:
3 ml litmus cream + 3 ml distilled water + 3 ml lipase solution

Test tube #2 contained:
3 ml litmus cream + 3 ml bile solution + 3 ml lipase solution

Both were placed in a 37 degree C water bath for an hour. When we pulled them out, both were a mauvish pink, and looked identical in color. Both solutions had definitely become more acidic, but seemingly to the same degree. (The litmus cream starts out bright purple but turns more red if the substance becomes more acidic and turns more blue if the substance becomes more basic.)

The question I have to answer is "Which tube showed the most hydrolysis?" My thoughts are that #2 should have been more pink/acidic since the bile would emulsify the fat in the litmus cream and the lipase could more easily hydrolyze it.

**Math Is Hard** · Mar9-06, 11:11 PM

No ideas, eh? Well, nevermind. I suppose we came to the right conclusions in the end - we received 100% credit for the lab.

**Ouabache** · Mar11-06, 08:02 PM

Originally Posted by Math Is Hard

We had a lab tonight involving enzymes. I am concerned about two of the results we got.

Test tube #1 contained:
3 ml litmus cream + 3 ml distilled water + 3 ml lipase solution

Test tube #2 contained:
3 ml litmus cream + 3 ml bile solution + 3 ml lipase solution

Both were placed in a 37 degree C water bath for an hour. When we pulled them out, both were a mauvish pink, and looked identical in color. Both solutions had definitely become more acidic, but seemingly to the same degree. (The litmus cream starts out bright purple but turns more red if the substance becomes more acidic and turns more blue if the substance becomes more basic.)

The question I have to answer is "Which tube showed the most hydrolysis?" My thoughts are that #2 should have been more pink/acidic since the bile would emulsify the fat in the litmus cream and the lipase could more easily hydrolyze it.

From your experience, did you know that lipase enables breakdown (hydrolysis) of fat into its components; glycerol and 3 fatty acids?

Why did you expect the resultant to be more acidic?
You are right to expect the one with bile to emulsify the cream (to smaller fat particles) and increase accessibility of each particle to the lipase.

Since both tubes appeared the same degree of acidity, how might you
change your experiment to see a difference between the resulting tubes?

[I can think of three ways]

**Math Is Hard** · Mar11-06, 09:04 PM

Hi Ouabache.

Originally Posted by Ouabache

From your experience, did you know that lipase enables breakdown (hydrolysis) of fat into its components; glycerol and 3 fatty acids?

yes, but not from the lab experience, just from what I read about the breakdown of fats prior to the lab.

Why did you expect the resultant to be more acidic?
You are right to expect the one with bile to emulsify the cream (to smaller fat particles) and increase accessibility of each particle to the lipase.

My thoughts were that both tubes would have a more acidic solution at the end of the test because of the hydrolysis of the fat in both cases. But, I thought that with the second tube, the bile would accelerate the process due to the emulsification, and the solution would increase in acidity at a faster rate than just the solution with just the lipase working alone.

Since both tubes appeared the same degree of acidity, how might you change your experiment to see a difference between the resulting tubes?
[I can think of three ways]

After I made the first post I went back and looked at some powerpoint slides about the lab that the instructor posted online. Sure enough, there was an image of what the results "should be" which showed tube #2 with a much redder (more acidic)solution. However, everyone in the class got the same result - both tubes were the same color. My guess was that maybe the tubes were left in the water bath too long and that's why we didn't see a dramatic difference. Maybe the cream had hydrolyzed as much as it could - with or without the bile emulsification? Perhaps if we had stopped sooner, we would have seen that tube #2 was much further along in the process?
Or maybe, we didn't leave the tubes in long enough, and enough time hadn't passed for either to show which solution was truly ahead in the hydrolyzing of the cream?
Do you think it could have been a timing issue --- or something else?
thanks.

**Ouabache** · Mar12-06, 02:23 PM

Originally Posted by Ouabache

From your experience, did you know that lipase enables breakdown (hydrolysis) of fat into its components; glycerol and 3 fatty acids?

Originally Posted by Math Is Hard

yes, but not from the lab experience, just from what I read about the breakdown of fats prior to the lab.

Very Good

Originally Posted by Ouabache

Why did you expect the resultant to be more acidic?
You are right to expect the one with bile to emulsify the cream (to smaller fat particles) and increase accessibility of each particle to the lipase.

Originally Posted by Math Is Hard

My thoughts were that both tubes would have a more acidic solution at the end of the test because of the hydrolysis of the fat in both cases.
But, I thought that with the second tube, the bile would accelerate the process due to the emulsification, and the solution would increase in acidity at a faster rate than just the solution with just the lipase working alone.

Great explanation

Originally Posted by Math Is Hard

After I made the first post I went back and looked at some powerpoint slides about the lab that the instructor posted online. Sure enough, there was an image of what the results "should be" which showed tube #2 with a much redder (more acidic)solution.

If the power point image indicates a more intense red color than the one you obtained, this suggests time was a factor. If you leave them in there for a much longer period and the color still says the same, you can make a reasonable assumption that the reaction is complete and the shade of red you see is as intense as it is going to get.

Originally Posted by Math Is Hard

However, everyone in the class got the same result - both tubes were the same color. My guess was that maybe the tubes were left in the water bath too long and that's why we didn't see a dramatic difference.

That is quite possible. How might you explain the difference between the color in your classes' tubes and the one on your powerpoint slide?

Originally Posted by Math Is Hard

Maybe the cream had hydrolyzed as much as it could - with or without the bile emulsification? Perhaps if we had stopped sooner, we would have seen that tube #2 was much further along in the process?

That's a good thought. How might you test this idea?

Originally Posted by Math Is Hard

Or maybe, we didn't leave the tubes in long enough, and enough time hadn't passed for either to show which solution was truly ahead in the hydrolyzing of the cream?

Another good thought. What kind of additional experiment(s) could you perform to resolve this?

Originally Posted by Math Is Hard

Do you think it could have been a timing issue --- or something else?
thanks.

Certainly time plays an important role in rate of enzyme mediated reactions. However there are several variables which affect rate-of-reaction (temperature, amount of enzyme and % active-enzyme as well as time). Manipulating any one of these variables (one at a time), can help you to resolve a difference in color between the two samples.

**Math Is Hard** · Mar16-06, 02:53 AM

If I had it to do over again, which unfortunately I don't, I think I would maintain tight, personal controls on the time and temperature, checking differences at 15 minute intervals.

After we did the initial mixing, we handed everything over to the teacher for the rest of the procedure, and since she was lecturing during all of this and not keeping a strict eye on the time and temp, I am sure these variables weren't precisely controlled.

I am glad you mentioned temperature - this was something that crossed my mind. Perhaps the water bath got too hot and denatured the enzyme?

Another thought: Increasing the amount of enzyme or bile (or both) in another sample might also possibly yield different results if we had been working with weak concentrations of these substances. Were you hinting at that? I am not sure.

**Bystander** · Mar16-06, 04:07 AM

http://arbl.cvmbs.colostate.edu/hboo...iver/bile.html

discusses "bile acids." New concept to me, but what's a chemist know; and,

http://en.wikipedia.org/wiki/Bile presents what I thought I knew about bile, "a greenish-yellow alkaline fluid...."

In either case, a proper experimental procedure is going to tweak the pH of the distilled water used in the first case to equal that of the bile solution used in the second. Otherwise, the pH indicator isn't "looking" at the same acid-base stoichiometry.

**Ouabache** · Mar20-06, 05:33 PM

Originally Posted by Math Is Hard

I am glad you mentioned temperature - this was something that crossed my mind. Perhaps the water bath got too hot and denatured the enzyme?

That could have happened (denaturation). It would explain why both sol'ns didn't acidify as much as expected.

Another thought: Increasing the amount of enzyme or bile (or both) in another sample might also possibly yield different results if we had been working with weak concentrations of these substances. Were you hinting at that? I am not sure.

Yes that is one of the parameters I described. So you might vary it to change the rate of reaction. Since the enzymes are released unchanged, after mediating a reaction, they will go work on other fat particles until all are broken down. With more enzyme in solution, you increase the rate of breakdown (more particles being worked on).

Bile is a different story.. It's salts are actually used in emulsifying fat. If you were doing this from scratch, you might design some experiments to determine if you were using too little bile. In your case, I would make a reasonable assumption that your experiment was designed so that a sufficient amount bile is present. Bystander makes a good point about adjusting the pH in both samples, because bile is already alkaline to start with. If your solutions were buffered, that would help keep their pH similar.