Cysteine / Cystine estimation in BSA (long(winded))

Emir Khatipov khatipovNO at NOuchicago.edu
Mon Oct 29 19:20:49 EST 2001


You probably understood already that it is not SH groups of you protein are
eluted more gradually compared to elution of the protein, but rather it is
BME that elutes after you protein. This BME reacts with DTNB, because it
also contains SH-groups. I did not clearly understand it from you message,
but I assume that the protein fractions did give you some OD412 values. Then
just use them for your calculations. Disregard the BME tail. Just determine
SH in your protein peaks. If you see no SH in your protein peak, then you
might have DTNB that is too old (should be prepared fresh), or BSA not
totally denatured, your buffer might be too basic, etc.

You can check at NCBI (
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=protein&list_u
ids=3336842&dopt=GenPept) that BSA has almost 30 cysteines per molecule, so
you should be able to see them with DTNB.

Emir


"Josh" <jj_barnett at froggy.com.au> wrote in message
news:3bd2e42c.0 at mercury.planet.net.au...
> Hi there. Sorry for the low-brow question, but I am struggling with a prac
> write-up at uni. Basically;
>
> 1) std curve of BSA in Guanidine HCl - Tris HCl by Bradford method -
> Absorbance at 595 nm
> 2) run reduced ( by beta-M.EtOH ) BSA through sephadex column, collect 12
> fractions, add DTNB and record absorbance at 412 nm
>
> The question the lecturer has set is;
>
> " Calculate the concentration of sulfhydryl group per unit mass of protein
> before and after reduction. Estimate how many moles of cystine are present
> in a mole of BSA."
>
> We are given the eqn,
>
> A 412 = E(DTNB).Cc
>
> E(DTNB) = 13 700 L/(mol.cm)
> Cc = concentration of cysteine reisudes
>
> OK, so I have my std curve, I also have set of readings by Bradford method
> from column fractions ie
>
> 1    0
> 2    0
> 3     1.10
> 4    0
>
> so the protein all came out in fraction 3, right?
>
> The problem is, the absorbances at 412 nm for the DTNB don't match up, ie
> peak absorbance builds up gradually to fraction 6 of 12, but doesn't
return
> to zero like the readings for Bradford method.
>
> The thing I am trying to get my head around is why the -SH groups that
react
> with the DTNB elute more gradually than the whole protein? That is, how do
I
> relate the concentration of cystine in BSA to the concentration of protein
> that came out of the column?
>
> Or are my results just shite?
>
> Note: previously in this prac we had to determine experimentally the molar
> absorptivity of tryptophan and tyrosine in 6M Guanidine HCl -Tris HCl, and
> then work out the number of tyrosine and tryptophan residues per molecule
of
> BSA. I did this after much headache, but this prac has got me baffled!
>
> Thanks for any pointers...:)
>
>
>





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