Qn on affinity column chromatography - regarding some of the mechanism behind it (Dec/08/2005 )

There is a question regarding the mechanism of affinty chromatography I like to ask. One way of eluting the protein of interest from the column is using a competitor eg. glutathione.

But in a way, reduced glutathione is more effective in eluting proteins. That goes my question, what is the mechanism behind the reduced glutathione that makes it more effetive than normal glutathion in eluting proteins ? Also, since there is a reduced form of glutathione, there must be a oxidised form of glutathione. Thus, why can't I use oxidised form ?

I hope someone can explain here in detail to my question as above.

Thanks in advance

Okay, I'm going to give this a try... though I cannot be absolutly sure of my answer, I am fairly sure of my answer.

If you reduce the disufide bond into a free sulfhydryl you get a SOH group instead of S-S.. so maybe that is more effective in eluting your protein off the column because the hydrogen is there.

The oxidized form (by adding Iodoacetimide) create an SO group. The hydrogen is no longer there to react with your protein on the column... and maybe that is important, though exactly how, I am not sure.

Rereading my answer... rather vague I know. Sorry.