The pH will govern this. Keeping the Henderson-Hasselbalch Equation in mind [pH = pKa + Log (base/acid), at a given pH, I would determine the distribution (deprotonated/protonated) of each acidic – basic group within the amino acid. The difference between the pH and the group's pKa is the log of the distribution of Base form / Acid form (or deprotonated/protonated) [see the HH equation]. So, let’s try glycine at pH 6.0: the amine has a pKa of 9.78. invLog (6 - 9.78) = 1.66E-4 deprotonated/protonated amine; the acid has a pKa of 2.35… so, invLog(6 – 2.35) = 4.47E3 deprotonated/protonated carboxylic acid. So, at pH 6, glycine is about .02% fully protonated (+), about 99.9% neutral (protonated (+) amine and deprotonated (-) acid), and about .02% fully deprotonated (-).

Thank you very much for your quick answer. This data can be obtained from distribution diagrams for amino acids, but I need percentage neutral (not-zwitterionic form). Please see left structure on the picture:

https://en.wikipedia.org/wiki/Zwitterion#/media/File:Amino_Acid_Zwitterion_Structural_Formulae_V.1.svg

Well, unfortunately the structure on the left essentially never exists. The Keq for that pair of structures would come from the two pKa's, and since each group on the left (at least at pH example) occur at .02% compared to the neutral, the left structure would only occur .02% of .02%. But unfortunately I think your question is a bit more complicated. There really are 3 forms, and the structure drawn on the left is unfortunately a 4th that doesn't occur. So I'm not entirely sure that reaction drawn is a meaningful reaction. Sorry I can't be of more help. This is certainly an interesting question.

I know it's a percentage very small, almost negligible because amino acids existing primarily as zwitterions at environmentally and physiologically relevant pH values in the bulk aqueous solution. But, why I asked? During formation peptide bond, amino acids can´t exist in zwitterionic form, because the protonated form of the amino group (ammonium ion) does not have nucleophilic properties. In each peptide-building reaction, the amino acids are presented in the neutral form. A similar situation we have during conversion alpha D glucopyranose to beta D glucopyranose and vice versa via aldehyde form (equilibrium mixture contains less than 1% aldehyde form). Thank you for your responses.

Regards.

The context for your question is helpful. If you want a Keq for the exact reaction shown in that diagram, then, treating it as a two step process, deprotonating the COOH and protonating the amine, then you would multiply the two Ka's to find the total Keq.

I agree, equilibrium expression for the overall reaction (shown in diagram) is equal to the product of the equilibrium expressions for the two reaction steps as you mentioned.

Dear Daniel, if you have a free time, please read please read these interesting manuscripts (something about this problem):

http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.781.2794&rep=rep1&type=pdf

http://www.pnas.org/content/109/39/15697

Best regards,

Milan.

Very interesting. Seems that there has been a great deal of work to find rather unusual environments to limit the presence of the Zwitterion. Presumably, amino acids in these unusual environments would have non-standard Keq's, so calculating these (predicting) might be challenging. Experimentally, approaches that evaluate their partition coeffients might be the simplest. Here is an example, although their approach might be more complicated than necessary. Thanks for sharing. This has been interesting to think about.

Best regards,

-Daniel

DOI: 10.1039/P29920000079

The partition coefficient seems interesting and easy method, but in this case, we have a new equilibrium between the water and octanol and it can disrupt equilibrium in water. I agree, this problem is not easy to solve. Maybe some spectroscopic approach? For example, we record UV spectrum (or IR, NMR) of an aqueous solution of alanine. After that, adjust the pH of the two units below the isoelectric point. If the absorbance (or some another signal) changes, this means that in the first solution the amino group is not completely protonated and that the difference is from the non-protonated amino group. Two (or more) pH units above the isoelectric point refer to the COO- group. Of course, the sensitivity of these measurements is questionable.

Best regards,

Milan.

Yes, spectroscopy may not give the precision you'd like. Perhaps a simple acid/base titration might tease out this information. I'm reminded of the titration approach to determine the basicity and alkalinity of water samples, you are able to determine the proportions of carbonate, bicarbonate, and carbonic acid in an aqueous sample.

Another idea that comes to mind is hydrogen/deuterium exchange MS. We had done a study a long time ago, observing the rate of deuterium exchange and the total amount of exchange. In typical HDXMS methods, no one cares about the rates, just the total incorporation, and these are with peptide or proteins. I've been considering that the rate and amount of incorporation, even with monomers ought to be a function of the amino acid form, and pH. Perhaps the info you're looking for could be teased out of that info. The isotope labeled MS data would give you the precision, if you can relate the either the rate of incorporation, and/or the deuterated /non-deuterated proportions back to the distribution of amino acid forms. Just some ideas that are coming to my mind.

Best of luck.

DOI: 10.1002/rcm.746

If I understood correctly question. OP is asking for experimental method to determinate equilibrium constant between two states of amino acid. Base on my experience( aspartic acid and polymers of it) where I examin binding efficiency of Amino acid with metal ion. I used Isothermal Titration Calorimentry, of course change pH as Dr King suggested above. I think this can be done by ITC... or much more expensive technique that can determinate above is High spead laser spectro...but I havent use it yet.

I would like to ask a similar question. Why and how creatine(zwitterion) cyclized into creatinine? The spontaneous non-enzymatic reaction of cyclization of creatine into creatinine depends on pH value of the solution and it is the fastest at pH=3, but this reaction occurs even at neutral pH (around 7, after the dissolution of creatine). This is one interesting manuscript in which authors were using Raman spectroscopy and detect neutral, zwitterionic and protonated species of creatine in solution. This process is very similar to peptide bond formation.

Thank you for your answers.

Best regards,

Milan.

Raman spectroscopic approach to monitor the in vitro cyclization of creatine → creatinine

doi: 10.1016/j.cplett.2014.11.021

Ask the authors directly, they are on Reserch gate. But they used DFT approach which mean they prove it theoretically

The answer to the question "Why creatine cyclized" is completely unknown.

The presence of water molecules seems to be crucial, probably due to entropy contribution. I start with experiments today, wish me luck.

Regards,

Milan.

How do you plan to start experiment without synchrotron?

First, we will do a simpler (and cheaper) measurement and try to determine how water molecules behave during cyclization. We will definitely do DFT calculations.