hydroxylamine cleavage in GuHCl

Roney Graf roney.graf at uni-konstanz.de
Tue Mar 24 07:54:13 EST 1998


   I am trying to cleave a protein at a unique N-G site using
hydroxylamine. Since the protein is poorly soluble (I have inclusion bodies
from an E. coli prep), I need to perform the reaction in GuHCl. There are
some references giving a simple protocol for this, but they seem to be
lacking some info. 

   Saris et al (Analytical Biochemistry 132/54-67/1983) say: Dissolve 1.1g
NH2OH-HCl (2M), 4.6g GuHCl (6M), 15mg tris (15mM) in 4.5M LiOH to get 8ml
at pH9.3. 

   The problem is, there is no way I get the NH2OH and the GuHCl dissolved
together. Separately, each of them dissolves to a cloudy solution which can
be clarified by filtration, but together it's just a mess. I'm using NaOH
instead of LiOH, but as far as I get it the LiOH is just used because it's
volatile and easier to get rid of. Or is it?

   So what's the deal? Does anybody out there have any experience with this? 

   Thanx in advance, all suggestions are appreciated!

roney



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