I have collagen type I dissolved in a 0.01M HCl solvent (pH 2), and need to exchange the majority (99%) of the H2O to D2O. I am thinking that dialysis against a corresponding 0.01M DCl D2O solution should work, but I wonder whether there is a risk that the collagen would escape through the membrane even if the size of the pores is small? My thinking is that the collagen would be denatured/have a loose structure and therefore could pass through the pores as "strings through a hole"...
Does anyone have experience with this or other suggestions for exchanging the H2O to D2O?
I think it is unlikely that collagen will pass through the pores in dialysis tubing with typical molecular weight cutoffs of about 10,000. Collagen is a very large protein.
Another way you might be able to do the exchange is with a gel filtration column equilibrated with D2O.
Or you could evaporate the water by lyophilization and rehydrate the sample with D2O.
Thanks, that is what I thought too. I will probably try the dialysis, but the other suggestions are also good.
Thanks!
Be aware that D2O is "hygroscopic", means that water from the air will dissolve in there, so keep the tubing closed well, eventually flush with Argon beforehand. Change D2O once to get the rest of H2O out. Quiet expensive dialysis, though :)
We usually order collagen type I from Roche as a solid, lyophilized material so you could choose in which solvent you want to dissolve it. I attached the link, may be interresting for you. It is for three bottles with 10 mg each, sterile packed.
www.sigmaaldrich.com/catalog/product/roche/11179179001
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