I really, really want to get isoelectric focusing...

[exparrot]

Hi everyone, I'm new to the forum and have this question on biochemistry related to a homework assignment that I can't wrap my brain around.

 

So I'm told that a mixture containing the following two polypeptides were subjected to an isoelectric focus gel electrophoresis. Two bands were observed. I need to identify which band belonfs to each peptide, and briefly explain why.

 

The polypeptides:

Thr-Ala-Asn-Phe-Glu

Pro-Gly-Thr-Cys-Met

 

I have a diagram which shows a band at around pH of 5-7 and another at pH of 9-11. I know this procedure utilizes the isoelectric point of a protein, but so far in my study of biochemistry (1 month into the semester), I've encountered the isoelectric point of amino acids. I'm not sure about proteins. Basically, I want to understand how can I figure out the isoelectric point of a protein, thus be able to tell which band belongs to which polypeptide. I would super appreciate any help!

 

If you (reader) intend to respond, please do it fast because I need this before 2:40 PM

[CharonY]

Fast responses are not our strength. It is odd, however, that anyone should explain IEF without explaining isolectric points (pI). There are a number of ways to do that (with varying accuracy) according to the pK of the amino acids. And right there is the hint you need. Second hint, the pI is the point where the net charge is zero.

That being said, just by looking at the peptides I find it unlikely that that one of the peptides is actually in the high basic range. Check up on the properties of the listed amino acids and you will see what I mean.

 

If you are interested in following that up, read Bjellqvist, B.,Hughes, G.J., Pasquali, Ch., Paquet, N., Ravier, F., Sanchez, J.-Ch., Frutiger, S. & Hochstrasser, D.F. The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis 1993, 14, 1023-1031.

 

Edit: I misunderstood the post. I assumed that you did not had heard about pI, but apparently you had it for AAs already. In any case the precise determination for whole proteins is a bit trickier. However, knowing the pI of the AAs will give you a good hint what to expect from the short peptides. One problem is, however, that the presented peptides are so short that most predictions will be off.

Edited October 1, 2009 by CharonY