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Topic: Protein purif  (Read 6802 times)

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Offline orgo814

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Protein purif
« on: September 14, 2014, 07:12:48 PM »
Question from hw was: a) In what order would the amino acids Arg, His, and Leu be eluted from a carboxymethyl column at pH 6? b) in what order would Glu, Lys, and Val be eluted from a diethylaminoethyl column at pH 8? Answer for a) Leu, His, Arg and b) Lys, Val, and Glu. My question is WHY

For A I figured leucine would be eluted first because it is non polar side chain which will not interact with the stationary phase. Histidine possibly next because it's pKa for side chain is 6.06 which means it won't interact as well with the negative charge on side chain followed by arginine with a higher pK

For B, I honestly would have that valine would go through first with the non polar side chain. But I'm assuming lysine first because the stationary phase is positively charged (I think) and lysine also pos charge will not interact, followed by valine, and Glu with its neg charge will interact with stat phase so it's last.

This is confusing me though I don't know if my reasoning is even right, and why the non polar would go through first in part A but not with part B

Offline Babcock_Hall

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Re: Protein purif
« Reply #1 on: September 14, 2014, 09:22:55 PM »
Diethylaminoethyl is a weak anion-exchanger, and it is positively charged near neutral pH.  I think it is generally helpful when doing these sorts of problems to recall that the net charge of an amino acid is dependent both on pH and its own pI.

Offline orgo814

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Re: Protein purif
« Reply #2 on: September 15, 2014, 09:02:13 AM »
So I'm still struggling with my question though. Why non polar wouldn't go through first like the first one etc

Offline Babcock_Hall

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Re: Protein purif
« Reply #3 on: September 15, 2014, 10:11:55 AM »
Does the nonpolar amino acid electrostatically repel the exchanger in the second question?

Offline orgo814

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Re: Protein purif
« Reply #4 on: September 15, 2014, 04:17:37 PM »
I'm gonna say yes, since the amino exchanger is polar charged. However there is also the ethyl groups? I'm not sure this is confusing me. How is my reasoning for the first problem if you don't mind me asking also

Offline Babcock_Hall

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Re: Protein purif
« Reply #5 on: September 15, 2014, 08:25:11 PM »
The dominant effect on ion exchangers is usually charge, which is not to say that hydrophobic interactions can always be ignored.  Hydrophobic effects are known with charged molecules which also an aromatic ring, for example.

In part (a) one might guess that aspartate or glutamate would elute first.  Amino acids with neutral side chains are tricky; their behavior is highly pH dependent.  For example, alanine will easily stick to Dowex 50 (a strong cation-exchange resin) at low pH.  The carboxylic acid is protonated, and so alanine binds via its positive charge on the alpha-amino group, which is obviously protonated.

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