hydroxylamine cleavage in GuHCl

Judith A. Airey jaairey at med.unr.edu
Thu Mar 26 14:41:24 EST 1998


I had similar problems with making the solutions.  By trial and error I
found that the following solution works.  The order of additions is
important, with the solution being right on the edge of its solubility.

For about 20 ml

LiOH 1.678g (2M)
add 8 ml water and stir.
GdHCl 11.46g (6M) add and stir until dissolved.
add NH2OH.HCl 2.78g (2M)

After stirring a couple of mins adjust to pH 9 using LiOH (or occasionally
HCl).  Be careful when you get close to pH 9 since there is little
buffering capacity at this pH.  Use immediately.

Hope this helps.

Judith



----------------------------------------------------------------------------

Judith A. Airey
Dept. Pharmacology/318
University of Nevada, Reno
Reno, NV 89557

Tel: 702 784 4651
Fax: 702 784 1620

On Tue, 24 Mar 1998, 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. 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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