Effect of DTT on protein structure/function independent ofdisulfide bond reduction

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Mon Mar 11 20:02:41 EST 2002


Buried disulphide ?! DTT will normally reduce those ONLY if you at least
partially unfold your protein... who knows if it will refold back.

Does your protein use metal ions ? DTT sucks out transition metals like a
sponge - to a lesser extent it also chelates alkali-earth metal ions.

In general, 50 mM is really high - if you can't reduce your disulphide by
lesser amounts (possibly would not murder your protein) then you should
consider trying something else - BME, TCEP, electrolysis etc.

Good luck.

A.G.E.
"Kartik Chandran" <kartik_chandran at hms.harvard.edu> wrote in message
news:B8B2B49D.942%kartik_chandran at hms.harvard.edu...
Hi there,

I'm investigating the effect of an engineered disulfide bond on the
hemolytic activity of the protein I'm studying. In order to reduce the
buried disulfide, I'm having to treat with 50 mM DTT at 37sC for 15-20 min
prior to addition of red blood cells (to test hemolytic potential of the
protein).

However, I get no activity in the presence of DTT even with a control
protein that lacks the two engineered cysteine residues. There are no other
disulfide bonds in the protein.

I'm wondering whether DTT may have effects on my protein independent of
reduction of the disulfide bond. If anyone has any ideas or input into my
problem, that would be great!

Thanks!

Kartik chandran

kartik_chandran at hms.harvard.edu


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