hydroxylamine cleavage in GuHCl

Scott McMahan mcmahan at oncology.wisc.edu
Tue Mar 24 19:11:30 EST 1998


In article <roney.graf-ya02408000R2403981354130001 at news.uni-konstanz.de>,
roney.graf at uni-konstanz.de (Roney Graf) wrote:

:   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.

What about using SDS?  I used it when I did a NH2OH reaction.  Look at
Biochemistry 33/12092-12099/1994

: 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. 

There's a better reference for the actual cleavage in Methods in Enzymology
#47.  I'm not sure of the pages, but the table of contents should tell you.

:   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?

The LiOH is used to prevent the precipitation of NaCl.  Since you're
starting with 8M hydrochlorides, the necessary NaOH to neutralize them
brings the total concentration of Na+ and Cl- pass the saturation point,
and you get a NaCl preciptate.  Filtering should give you a workable NH2OH
solution (the Li+ and Cl- ions are just spectators), as will using LiOH
(LiCl has a higher solubility) or using SDS as a denaturant instead of
GuHCl (less NaOH needed to get the pH up to 9.3 and only 2M Cl- from the
NH2OHHCl.

-- 
                                         Scott McMahan
                                         mcmahan at oncology.wisc.edu



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