Watch out with oils and oxygen. Oxygen is fairly soluble in oils. When we
worked on deoxyhemerythrin, we had to degas our sealing wax to keep things
anaerobic. If you heat wax, and then put it in a vacuum, it'll froth as all
the gases come out of the liguid. Then of course it hardens and you have to
heat and vacuum again. It took several cycles of that to degas the sealing wax
for our capillaries. (That shows how long ago that was.) Ron
On Thu, 2 Oct 2014, Boaz Shaanan wrote:
Hi Tatiana,
Your problem is most reminiscent to the problem that Max Perutz faced when he dealt with deoxy-haemoglobin crystals, but in those days only mounting in capillaries was the way to bring the crystals to the beam, so he used dithionite, just like you, but mounted the crystals in (specially made for him at LMB) glove box under Nitrogen atmosphere. I guess you're now freezing your crystals for data collection, right? I'm not sure how to do this in a glove box nor am I sure whether after freezing your crystals is protected against oxidation. Maybe. But perhaps you can also consider using Parthon oil (or something similar) as cryo-protectant so it will "coat" your crystal in the glove box and will also reduce oxidation?
Good luck,
Boaz
Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel
E-mail: [email protected]
Phone: 972-8-647-2220 Skype: boaz.shaanan
Fax: 972-8-647-2992 or 972-8-646-1710
________________________________________
From: CCP4 bulletin board [[email protected]] on behalf of ISABET Tatiana
[[email protected]]
Sent: Thursday, October 02, 2014 11:22 AM
To: [email protected]
Subject: [ccp4bb] Question about enzyme behavior
Dear all,
Sorry for a non purely crystallographic question.
I am working on an enzyme which binds Fe2+ cations to catalyzes an
FeII-dependent hydroxylation reaction.
Because of fast oxidation in presence of the enzyme, it is very difficult to
soak Fe2+ ions into the crystals. We succeed only under anaerobic conditions
(glove box). I use a combination of dithionite as a reducing agent and Fe2+SO4
or (NH4)2Fe(SO4)2 as Fe2+ source. Despite these precautions, the Fe2+ is most
often disordered in the active site.
When I add Fe2+ under aerobic conditions, Fe2+ oxidizes immediately upon
contact with the protein solution (despite 1mM Dithionite for 5mM Fe2+ and
protein concentration = 230uM). Furthermore, the hydroxyl donor molecule, which
should bind Fe2+ (before the substrate) and one residue of the protein, is not
seen in the electron-density maps in the active site. I have tried several
soaking conditions. When I try a co-crystallization approach, adding Fe2+ and
this hydroxyl donor molecule directly to the protein solution under anaerobic
conditions, the protein precipitates.
Does anybody have an idea or experience with this type of results? or how to
fix the molecule to such a site? What type of phenomena could occur at the
active site preventing the binding of the product?
Thanks for your help
Best regards
Tatiana
