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

Josh jj_barnett at froggy.com.au
Sun Oct 21 09:50:08 EST 2001


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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