thunderfvck
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Counting the charge on protein sequences
Hey there
Long time no see! I've been kind of busy lately, going to university now which brings me here. I have a question about one of these questions I'm
having trouble with.
It's for one of the labs we've done. We took pig LDH (heart and muscle - isozymes) and we put it in agarose gel, then we ran a current through it and
the isozymes (stained) separated. The heart isozyme moved while the muscle did not.
One of the questions for the report...
We had to blast the sequences of LDH-H and LDH-M for pig. The question I'm having trouble with reads: Now consider the amino acids that are different
between the two sequences. At pH 8.6 which of the two polypeptides (heart or muscle) will have a more net negative charge?
I highlighted the parts of the sequences which were different. Then I counted how many cysteines, histidines, aspartates and glutamates there were; I
counted these because their pKr's are all below 8.6 and so a pH of 8.6 will give them a net negative charge. I came up there being more negative
charge in the muscle than in the heart. The results show that the heart moved (so there must have been more negative charge in the heart isozyme than
the muscle)...So I guess I need to take into account the pI's as well. What I have to do then, is to count all the residues that have a pI below 8.6
and whichever polypeptide has more, has a more negative charge...right? How then do I take into the account of the pKr's? Do I simply count the pI and
neglect the residues with ionizable side chains?
I'm not sure I understand how counting the charge works. I found a protein calculator online which allows me to input the sequences. It gave me the
heart with more negative charge (which my results have shown)...and they took into account both the pKr and pI's. How is this done?
Thanks for any help anyone here can give me!
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chemoleo
Biochemicus Energeticus
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Do you know expasy.ch? They have a vast array of tools. If you check their help sections for a given calculator, you should be able to look up which
references were used to calculate the pIs.
In principle it could be just the average of all the different amino acid pKa's, although I suspect todays algorithms are a little more complex than
that.
Nonetheless it is a theoretical pI, as it assumes the sequence is unfolded where all the amino acids are exposed. In reality, with a globular protein,
some of the charges are hidden in the hydrophobic core, and thus do not affect the overall isoelectric point pI of the protein.
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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thunderfvck
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Thanks for the reply.
I went to expasy.org and couldn't really find anything of use...
Perhaps I wasn't clear. I don't need to CALCULATE the actual charge for a given sequence, just be able to say which one will be more negative.
Here's a bit of the alligned sequences:
DQLIHN (LDH-H)
++LI
EKLIAP (LDH-M)
So, to see which one has a more negative charge I will look at the differences in the sequence. This will be:
DQHN (LDH-H)
EKAP (LDH-M)
The pH of the buffer used to make the gel was 8.6. All the amino acids have pI's less than 8.6 except for lysine (K) and arginine (R) which are 9.74
and 10.76 respectively. So this means that at 8.6, lysine and arginine will be postively charged. It then follows that all the other amino acids
EXCEPT FOR lysine and arginine will carry a negative charge. Since LDH-M has the sequence EKAP and LDH-H has DQHN, LDH-H will carry a
more negative charge for this particular sequence because it contains one postively charge lysine while the other isozyme doesn't have any.
Am I correct in my reasoning?
Should I just worry about the pI's when estimating the charge of a protein?
Also, we are to assume that the "pKa's of the side chains are the same as those for the free amino acids" so protein folding isn't taken into
consideration.
Thanks a lot chemo!
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