Hydroxylamine protocol

Mark Brandt, Ph.D. mark at indy-lv.biomol.uci.edu
Fri Nov 8 19:41:40 EST 1996


In article <55kqei$qq3 at decaxp.harvard.edu>, remeans at fas.harvard.edu (Robert
Means) wrote:

> Hello,
>         Despite the Harvard U. in my identifier, I'm out in the woods
> without great library service. I need a protocol for hydroxylamine
> proteolytic cleavage. I know it should be in high pH, but I need a few
> specifics. Can someone out there e-mail me a protocol, or even FAX it if
> you have a paper copy and don't feel like typing it in. In the meantime
> I'm going to submit to our library service and wait for them to FAX me the
> pages I need next week. Yuch!!
> Thanks,
> Bob Means
> FAX (508)624-8190

It depends somewhat on whether you want the protein to survive the
procedure in some semblence of native conformation or don't care.

I use a method adapted from:
Bornstein P, Balian G (1977) Cleavage at Asn-Gly bonds with hydroxylamine;
Meth Enzymol 47: 132-45.

Final concentration:
2 M hydroxylamine-HCl
0.2 M Tris-HCl, pH 9.0

I make up a stock solution of 4 M hydroxylamine-HCl, 0.4 M Tris-HCl, pH
9.0. In order to obtain the correct pH, it is necessary to dissolve the
hydroxylamine in 5 M NaOH (you will probably need to adjust the pH slightly
afterwards). I then mix the hydroxylamine solution 50:50 with the protein
solution.
I perform the reaction at ambient temperature for 48-96 hours (depending on
protein of interest).

If you don't mind denaturing your protein, cleavage occurs much more
rapidly (complete in less than 8 hours) at higher pH (pH 10 may be optimum)
and higher temperature (42°C).

I hope this helps.

-- 
Mark Brandt, Ph.D.
My opinions are my own, but I tend to give them away to anyone who fails to
flee fast enough.



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