Sketch the change of pH in the breakdown of proteins into amino acids

[FlyingWhale]

Homework Statement

Trypsin is an enzyme that catalyses the breakdown of proteins into amino acids. 9cm³ of liquid egg white (protein) was mixed with 1cm³ of 1% trypsin in a small beaker. The pH of the solution was measured using a pH probe for 30 minutes.

The pH at the start of the experiment was pH 7.5.

On the axis below, sketch a graph to show how the pH would change over the course of the experiment.

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Relevant Equations

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The pH stays the same because the optimum pH of trypsin is around pH 7.5. I think??

 

[Borek]

Products of the reaction will change pH, unless the solution is buffered.

Not that I know what the changes will be. Could be I am wrong, but I feel like it should depend on the amino acids present in the egg white (which is definitely something known, just not for me).

 

[epenguin]

The optimum pH is not a cause of its staying the same. It will stay the same if the pH is not optimum and the trypsin does not work so well!

To a first approximation I would think the pH would not change, regardless of the aminoacid composition of the egg white. The trypsin catalyses only the hydrolysis of only alpha amino peptides, i.e.
...-NH-RCO-NH-R'CO... -> ...-NH-RCO₂^- + NH₃⁺R'CO₂...
The reaction at neutral pH produces or consumes consumed no net protons gives essentially no pH change.There might be a secondary effect due to a few amino acid side chain groups by themselves having a different pK than those same groups when they are part of the intact protein. There might be some difficult calculation to make about this, but anything like that anything like that is surely not expected in a question where they do not even tell you the concentration of your substrate protein!.

However it is worrying enough I would be glad if you would post the answer you are given when you have been.

 

[Borek]

@epenguin Hydrolysis doesn't consume nor produce H⁺, but it introduces -COOH and -NH₂ to the solution (you wrote them as -COO^- and -NH₃⁺ in your reaction equation, which is essentially the same thing) and that will definitely change the pH. Overall change will depend on pKa/pKb values, so the identity of amino acids sounds like an important factor.

I agree there is not enough information for the quantitative analysis, but perhaps there is enough data for a qualitative answer.

 

[epenguin]

I have to recognise that my previous answer should be considered misleading, though I don't think for the reasons that Borek says.

It is true that that a peptidase hydrolysate of the protein would contain almost 100% zwitterions NH₃⁺RHCO2^-, other forms hence being negligible for most practical purposes one might think. It's just that these practical purposes do not include our present one!

The reason is α-amino acids are weak bases (pK_a of their -NH₃⁺ group about 9.3) while the carboxyl groups are rather strong acids with average pK_a around 2.2. Considerably farther from 7. so a hydrolysate, mixture of all the amino acids, should be somewhat acid.

And that although the number of protons contributed to solution by this factor is small, the total number of protons in near neutral solution is small anyway, so the difference this factor makes in terms of pH can be considerable!

One can do some useful crude calculations. I think they are helpful or indispensable in order to understand the problem. Student is invited.

Fresh egg White has a pH of 7.5, as per the problem. Whatever else is there, it is actually mostly protein. Its concentration is around 0.1 g/ml. Take a very crude value of 100 for the average amino acid molecular mass, then that is an approximately 1M solution of amino acid. So calculate firstly what is the pH of 1M amino acid solution if all the side chains are neutral and the carboxyl pK_a's an average 2.2.

After which we will have to consider the side chains and their buffering effect

 

[epenguin]

More oversights on my part.

To say that a mixture of amino acids with pK_a 2.2 created by hydrolysis of proteins is almost totally in the zwitterionic form is not even a very good approximation. Though the calculation suggested was so crude this doesn't matter very much. It is not asking much of the student to calculate what the pH is for a 1M solution, he or others might be surprised.

That is just useful background. In fact trypsin does not reduce protein to amino acids (the thread title is wrong) but to peptides. To think further we need to take a relevant example. The best would be ovalbumin (42.7 kDa, 385 amino acid residue) which constitutes 50% of egg whites which we could take as typical of all protein content.

Trypsin cleaves the peptide bond between the carboxyl group of lysine or the carboxyl group of arginine and the amino group of the adjacent amino acid. There are 20 lys residues and 15 arg, so the protein is cleaved into 36 peptides on complete trypsin hydrolysis. What would happen to the protons liberated by this? You have to look at other buffering groups on side chains.

The relevant ones are his, pK_a≈6 of which there is one Per molecule ovalbumin, glu, pK_a≈4.25 of which there are 52 and asp, pK_a≈3.65. And then, so far as I can make out, there are two phosphoseryl groups, pK_a=7. What is going to happen to the protons liberated by peptide bond hydrolysis?

For exactly what happens in the experiments we would also need the pH/activity information which is has not been given. I would be chary of using some literature data because these things are often measured with (chromogenic) ester substrates, which I think give a pH-activity profile different from peptides. But we can qualitatively know.

in case you are thinking this is all too easy, I remembered that trypsin digests itself as well as the protein you wanted to. but I read that you can get a modified trypsin that doesn't do that so let us worry about one thing less.

 

[epenguin]

This student posted this question and a feeble illogical answer attempt 10 days ago and never came back since. It happens here sometimes, and usually we ignore such visits.

The question is in one way academic. You would normally perform a trypsinolysis in a buffer. However the experiment described seems perfectly feasible. and as academic questions go, I think it is a good and useful one. except for the lack of information supplied, such as the pH profile of trypsin activity.

What is useful, far more than the question in itself, is to be able to confidently answer questions like this. Or to know what information you need to be able to answer them. Students at this stage should be able to answer the question I asked in #5. Should be able to answer the question too: if the trypsinolysis goes to completion, what would be the pH at the end? I have given information you need for an answer.

I found I was, as maybe shows, a bit rusty and had to think harder. I think that, starting at certain pH at least, and especially if there are two phosphoryl groups in ovalbumin, its time evolution can be quite surprising at first sight because of the effects of buffering.