From: Richard M Thomas <rthomas@ifp.mat.ethz.ch>
  To  : RASMB@BBRI.HARVARD.EDU
  Date: Thu, 31 Jul 1997 13:52:15 +0200

Boiling GuCl

RE: Olin Spivey's question on the use of boiling samples in 6M GuHCl to
ensure protease denaturation.
	Samples are boiled in SDS solution in order to ensure full denaturation
and to speed the process up. While some proteases are remarkably resistant
to denaturation it would count as extremely unlucky to have one active in
6M GuHCl in your sample. Boiling wouldn't help if you did, as dogma
suggests that the protease would probably refold as soon as the temperature
was lowered back to the measurement conditions. If you have good evidence
for specific proteolytic activity there seem to be two answers (i) purify
the sample or (ii) add a specific inhibitor. Protease activity arises from
two sources - (a)contamination or co-purification and (b) the presence of
microorganisms. Boiling might help in the latter case by killing the little
..... Addition of bacteriocides can be of use here, too. Low concentrations
of azide are OK providing that you do not want to make optical absorbance
measurements below about 240nm (see recent correspondence from Arthur Rowe
and others).
Other protein contaminants can produce interesting effects - Don't do urea
denaturation experiments in the presence of ureases, and so on.
Hope this helps
Richard
Richard M Thomas
Inst. für Polymere
ETH-Zentrum
CH-8092 Zürich
Switzerland
(tel) +41 1 632 5540
(fax) +41 1 632 1073