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Topic: Indicator Test for Protein Hydrolysis Experiment  (Read 1070 times)

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Offline WeezyWeezy

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Indicator Test for Protein Hydrolysis Experiment
« on: March 12, 2023, 09:15:48 PM »
Using egg white powder solution for protein and adding various forms of ginger (contains zingibain protease enzyme). But I need a test to check how active the enzyme has been.

I originally was gonna use a biuret test, and tried a few times using sodium hydroxide and copper sulfate. It worked, except there was no difference at all between my control (just powder) and ones which had ginger added.

I then did some more research and found that the biuret is nowhere near sensitive enough to indicate differences in the presence of peptide bonds in such small quantities. What test can I use instead?

My school doesnt have any protease reagants already made up, so it needs to be something I can make myself with the chemicals themselves (I did this for the biuret test, no problem). Or is there another way i could check how much hydrolysis has occured that doesnt involve a reagant test?

Keep in mind that I will be using a colorimeter to quantify colour changes (dont have a spectrophotometer at the school).

I'm really stuck please help.

Offline Babcock_Hall

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Re: Indicator Test for Protein Hydrolysis Experiment
« Reply #1 on: March 13, 2023, 09:32:55 AM »
I am not yet convinced that the principle of your assay is correct.  If a protease acts on a polypeptide, the products are shorter peptides.  The shorter peptides may still give a positive biuret test, but I am not 100% certain. 

Offline Corribus

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Re: Indicator Test for Protein Hydrolysis Experiment
« Reply #2 on: March 13, 2023, 01:00:37 PM »
Seems zingibain is a specific protease, meaning it only cleaves at certain residues, in this case proline. (Link) This is contrasted with a protease like trypsin that cleaves indiscriminantly.

With that in mind, I agree with Babcock Hall. Protease activity here is just going to give you smaller peptides, so the biuret test by itself will probably not be effective.

If you can get your hands on some filters with defined pore sizes, you might be able to take advantage of the fact that the parent protein is larger than the cleavage products. I.e., find a filter where the pore size is smaller than parent proteins but larger than the products. If you force your mixture through the filter, any parent protein will not go through.

EDIT: You may also want to consider whether this protease is effective with egg albumin. First requirement is that albumin contains proline (it does, which you can see by checking the protein databases, e.g. here). But whether the proline is accessible given the protein's native 3D structure is also a consideration. Maybe is, but just something to think about.
What men are poets who can speak of Jupiter if he were like a man, but if he is an immense spinning sphere of methane and ammonia must be silent?  - Richard P. Feynman

Offline Babcock_Hall

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Re: Indicator Test for Protein Hydrolysis Experiment
« Reply #3 on: March 14, 2023, 09:11:35 PM »
One way to follow a protease reaction is to perform sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) on the reactant and products.  The products will move more quickly through the gel, being smaller.  This is a common experiment at the junior/senior level of undergraduate biochemistry, but it is not practical in all situations.

Offline Babcock_Hall

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Re: Indicator Test for Protein Hydrolysis Experiment
« Reply #4 on: March 15, 2023, 05:42:17 PM »
BTW, trypsin is selective for positively charged side chains, lysine and arginine.

Offline Corribus

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Re: Indicator Test for Protein Hydrolysis Experiment
« Reply #5 on: March 16, 2023, 11:07:11 AM »
Sorry, you are correct. I remembered only that it was less selective than a lot of other proteases and wrote that without checking. Thanks for pointing that out!
What men are poets who can speak of Jupiter if he were like a man, but if he is an immense spinning sphere of methane and ammonia must be silent?  - Richard P. Feynman

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