Ph Effect on enzyme in digesting protein

[margaret collord]

We're trying to figure out what we're doing wrong- and we're running out of time.
Project= Effect of PH on enzyme in protein digestion. We usd pineapple juice as enzyme + added either HCL, base, or water. we made 2 concentrations of each starting with 1 oz of pineapple juice (canned), about 50 drops of acid (one with 25 drops hcl, 25 drops water), base (same two concentrations as the acid I just mentioned) and last one with 50 drops water. we added a piece of cooked egg white as our protein (about 1/2" x/1/2" x1/4" size piece)- 2 days later no readable result.

Cut the samples to just 3 (vs the original 5) and after adding 70 ml of Hcl into 1 (and an addl 70 ml of either ammonia or water to the corresponding other mixtures) we did we get reaction after 6 days (added extras on day 2).

Not happy with result (due to adding so much additional acid) we've tried again after more research. This time we used hardened gelatin as protein - about 30ml in bottom of petridish and added 3ml FRESH pineapple juice (vs canned) + 1ml of the acid, base or neutral. 2 hours later no visible result. 2 days later- result not as expected. Base + neutral are thin liquids, acid is liquid too but thicker than the others suggesting it is the slowest to digest? this doesn't make sense to me.

Can you suggest better way w/quickly observable result? I've read that Pepsin might be a better enzyme-but I don't have time left to order by mail and I'm not sure OTC supplements yield the right result. We started this project with plenty of time, but now we're behind the 8 ball - its due on 3/1.

[deleted-42343]

Fun project! And very timely I might add, since a project very similar to yours was just demonstrated on TV yesterday .

You could try meat tenderizer (from the grocery store), which contains the enzyme papain. Here's the wikipedia article about it:
http://en.wikipedia.org/wiki/Papain

We have a project very similar to this, but you could try putting the meat tenderizer into acid or base first, and then add it to the gelatin.

Here's the project idea: https://www.sciencebuddies.org/science- ... p004.shtml
There's even a video about this project that was on TV yesterday: https://www.sciencebuddies.org/blog/201 ... essert.php

As for your results, I definitely think using gelatin will make everything easier, as it will obviously be liquid or solid. The egg would have been harder to see.

You have to keep in mind that each kind of protease, and proteins in general, are built for a particular range of pH values. Some will be fine in acidic solutions, and others will not work properly in basic solutions. Some have a large range of values they can be in, others are very sensitive in changes in pH.

I also would have thought that the acidic pH would have allowed the protease to work better (as fruit juice is acidic), and thus make the jello more liquid than the basic solution.

Are you sure the concentrations of your acids and bases are the same? What base are you using?

One idea is that since the fruit juice is acidic and you added additional acid, that the environment was too acidic for the protease and it was not working as effectively. The fruit juice would have counteracted the base you added as well. So maybe the more basic environment was a bit more ideal than the acidic environment.

It's also possible that for whatever reason, the jello just did not set properly, or that the jello wasn't mixed enough. I would do at least 3 cups (i.e. 3 trials) of jello for each pH to ensure your results are accurate.

Good luck and let us know if you have more questions!

[margaret collord]

thanks for our speedy reply. your reply is interesting _ I hadn't realized that making the solution too acidic could result in the opposite result. I'm going to have to give some thought as to how we can be rue to the problem, and have a clear answer as to which direction the pH change has the greatest effect. I was a little concerned starting with pineapple juice because it is an acid - but I had assumed that just adding a higher or lower pH to the solution would just increase/decrease the pH overall and give us a clear winner.

If we use meat tenderizer instead do you happen to know if that has a pH significantly off from neutral? Would I add it to water before adding it to the acid or base? any idea n how much I should use?

Our base is household ammonia. the label has no concentration listed - but deionized water is the first ingredient. (BTW Our HCl is 31% solution)

I have been approaching this from a point of view of breaking down the hardened gelatin (or cooked egg white) because it is my understanding that the enzyme will eventually break down the protein in all 3 different pH levels- just that one would be faster than the others. Some of the experiments I've found on line suggest adding the enzyme and acid/neutral/base when DISSOLVING the gelatin before it hardens- then watching it for 30 mins to see if any solidify. that seems backwards logic but i'm despirate enough to give it a go if it makes sense. I really enjoy the science fair with my kids each year. Love doing these experiments together. This year my daughter has a partner so I'm pretty frustrated that this isn't going smoothly - one thing to effect our own kids grade - whole other thing to affect another kids!

[deleted-42343]

You don't need to worry whether the enzyme itself will make the solution acidic or basic.

I think dissolving the gelatin with the meat tenderizer would be the easiest way to analyze results. It will either stay a liquid or solidify, and would probably give you faster results, too.

I just checked online and the pH of household ammonia is supposed to be about 11.

Your HCl solution is VERY strong. In order to get your HCl to be pH of 3, you would need to dilute it by adding 1 drop of the 31% solution to about 2 cups of water, and that should be about pH of 3. If there is a science store near you, you could even buy some pH strips to verify the pH, but this is not necessary. This way, both the base and the acid solutions are 4 away from pH of 7.

You should just use the ammonia as is. Sadly, with the HCl and ammonia in there, you won't be able to eat it . Maybe you can make more regular jello at the end .

You could add the meat tenderizer to a little bit of the acid and base solutions (so it is dissolved), and then add the gelatin and water like the project idea mentions in the procedure.

If nothing happens, you can try increasing the concentration. If you have time, you could even try and see if lower concentrations affect it, so for example, you could dilute the acid and basic solutions by 1 cup of water, then 2 cups...etc.

The enzyme can work outside of its pH range, and if this is the case, the reaction would be slower. However, I think if the pH is way too high or too low, it would shut down completely (denatured).

As a last resort:
If for some reason you just can't get the pH to work, you could try dissolving the meat tenderizer in some water and heating one cup and freezing another, and of course having one cup with room temperature tenderizer. Heat would denature the enzyme and it wouldn't cut up the protein (so the jello should solidify fine).

Let me know if you have more questions.

[donnahardy2]

Hi,

Amber has given you excellent advice on this project. This is an interesting experiment, and since you are at a deadline for writing up the result, you just need to explain why the fresh pineapple juice did not break down the cooked egg/ gelatin and perhaps give some ideas for your next experiment.

The enzyme in fresh pineapple juice is called bromelain and is a mixture of proteinases that break the bond between amino acids in the peptide chain of proteins such a gelatin. If you used fresh pineapple juice, then it should have contained active bromelain. Did you prepare the pineapple juice yourself, or did you purchase it? If it was purchased, is it possible that it was pasteurized before you tested it? If you can confirm that you had active bromelain, then the conditions were not optimum for gelatin breakdown. Here is a paper that reports the optimum pH and temperature for bromelain;

http://www.ias.ac.in/jbiosci/dec2003/709.pdf

Here is the Wikipedia website that gives slightly different temperature and pH values.

http://en.wikipedia.org/wiki/Bromelain

According to the paper with experimental results, the optimum pH is 6 and the optimum temperature is between 40 and 60 degrees Centigrade. What temperature were you using? As Amber mentioned, it sounds like you added a lot of acid and a lot of ammonia to two pH-extreme samples, so this would have put the pH out of the optimum range for these two samples. So the question is why the water diluted sample did not work. Did you try a sample of undiluted pineapple juice? Did you measure the pH of the water-diluted sample? Perhaps the pH of this sample was not optimum, or maybe the water diluted the enzyme too much? What kind of water did you use to dilute the pineapple juice? Perhaps it contained an enzyme inhibitor. There are a number of possibilities here that you can include in your conclusion section.

Don’t worry about getting results at this point. Just concentrate on writing up your results and explaining the science behind your project, and the possible reasons that the enzyme did not work.

Donna Hardy