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pI's of 2 proteins

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pI's of 2 proteins

Postby biology_06er » Tue May 05, 2009 4:08 am

Hi there,

We did an experiemnt the other day where we had to sep. out two proteins via a colum containing CM beads. It had a pH of 6 and both proteins bound to this. So to sep them out we used a pH of 7.6 and collected a sample and then used a pH and of 9 and collected sample 2. So now I have to comment on the charge properties and likely pI's of (cytochrome C and haemoglobin)..

CM contains a negative charge right? So that means in order for those 2 proteins to bind to it they both proteins must contain a positive charge?...and it says "for a given protein it is necessary to carry out ion-exchange chrom at a pH aboive or below its pI. Above its pI a protein will be -ve charged and above its pI it will carry a +ve charge"...

so if i was to say that both proteins carried a positive charge would this be correct? How would I work out what the pI is of each protein?

Thanks,
b_06er
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Postby mataiquang » Wed Mar 23, 2011 10:53 pm

HI there, im assuming your doing biosci 350?

Heres some info that i found which may help explain some stuffs,

its basically the same for haemoglobin just at a different pI.

Cytochrome c has a very high pI (9.52) due to the high ratio of basic to acidic residues on the molecule. A cation-exchange column, specifically carboxymethyl (CMC) is used to help purify cytochrome c. This is because the pH for chromatography is below the pI of cytochrome c, thus leaving the molecule with an overall positive charge. This positive charge allows it to bind to the CMC column, which has a negatively charged resin. When the time comes to retrieve cytochrome c from the column, the pH of the elution buffer has to be increased to a pH above cytochrome c's pI. This will make cytochrome c have an overall negative charge, and it will no longer be bound to the negatively charged resin in the column.

Hope this helps
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