Bicarb at low pH

From: Jacob Keller
Date: 2 October 2011 21:56

Dear Crystallographers,

I would like to soak my crystals in bicarbonate (a possible
substrate), but the crystals have grown--and only grow--in pH 5.2-6.0,
so the bicarb/CO2 will just keep evolving out of the solution and
reliquishing its hydroxyls until the pH is elevated sufficiently out
of range. Does anyone have a clever way of getting bicarb into these
crystals? Grow them under CO2? Transfer them to higher pH, and hope
for the best?

Jacob Keller

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From: Roger Rowlett

You can't change the pKa of CO2, which is 6.3. Any attempt to grow bicarb complexes below pH 7.5 will be problematic due to CO2 bubble formation, which may crack the crystal. What we do in these situations is to soak crystals in a cryo solution at a higher pH for as long as practical, then transfer to another, identical solution with bicarbonate fo li and soak. This can be tricky, because crystals may take a long time to pH equilibrate, crack, or dissolve. At pH 7.5 or above, CO2 formation at chemical equilibrium is minimal. At low pH, you can also consider using acetate as a bicarbonate analog.

Roger Rowlett

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From: Edward A. Berry

Assuming they grow by vapor diffusion- how about:
Replace the reservoir with diet coke (Perrier water
if you want to be high class) with pH adjusted to
the range you want and sufficient PEG or glycerol
to give the same osmolarity?
CO2 diffusing in will lower the pH but not much below
that of the reservoir. When you open the well to mount,
especially with hanging drops, the CO2 will leave
and pH will rise, but not above the starting point.

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From: Bosch, Juergen

You might want to consider misusing the Xe-chamber which a couple of labs and synchrotrons bought in the late 90's to derivative crystals with Xenon. Instead of flushing the chamber with Xenon you could flush it with CO2 then plunge freeze your crystals within the chamber.

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Jürgen

......................

Jürgen Bosch

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From: David Schuller

Is it the bicarb you are interested in, or the CO2? Domsic, et al were able to trap a carbon dioxide in carbonic anhydrase II by adding CO2 during a high pressure cryo-cooling experiment.

J Biol Chem. 2008 November 7; 283(45): 30766–30771.

doi:  10.1074/jbc.M805353200

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From: Richard Gillilan

Hi Jacob, high-pressure cryocrystallography methods may be useful in this case. I copied your question to Chae Un Kim here at MacCHESS and he forwards this suggestion:

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

Hi Richard,

 

I think pressure cryocooling might be useful.

 

They may want to check the following reference.

 

JF Domsic et al (2008), Entrapment of carbon dioxide in the active site of carbonic anhydrase II, J. Biol. Chem. 283, 30766-30771

 

Best regards,

chaeun

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From: Ed Pozharski

Consider crosslinking your crystals with glutaraldehyde.  They will then
became virtually insoluble, although it's possible that you may lose
diffraction at elevated pH.




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From: Mathews, Irimpan I.

Hi Jacob,

Once I had an issue with introuding CO2 into crystals and I was able to get it bound by putting a small piece of dry ice into the well solution and keeping the drop under CO2 pressure using nextal trays with screw cap.  Please note in this case CO2 didn't get bound by using pressure cell.  This could imply that CO2 gets incorporated into these crystals during crystal growth.

regards,
Mathews