[jrev005]

Hi all,

I have done surface staining studies of a transport protein, using an antibody which binds an extracellular epitope.
Binding of this antibody is dependent on an S-S bridge between two cysteines to be intact. If this is disrupted the conformation of
the epitope is changed and it stops binding. I have found that the compound, curcumin, and a monocarbonyl analogue with an a,b-unsaturated carbonyl group
could decrease cell surface immunostaining using the mentioned antibody. I would normally interpret this as a downregulation of the transporter at the cell surface
caused by curcumin and my test compound. However, I have read somewhere that curcumin and a,b-unsaturated carbonyl groups can act as michael acceptors and react with sulfhydryl groups. Is there a chance that my tested compounds do not infact downregulate the surface expression of the transporter and merely disrupt the binding of antibody to the transport protein by interfering with the S-S bridge? Possibly covalently binding and modifying it?


Thanks from a non-chemist,


curcumin structure

[Babcock_Hall]

I am a little bit confused.  Sulfhydryl groups can be nucleophiles in Michael additions.  However, disulfides, which are more oxidized, are not very nucleophilic at all.  If one of your proteins has a sulfhydryl group elsewhere, I would not be surprised to see a reaction.  I don't know how quickly the reaction would occur, however.

[discodermolide]

What is the staining method you used?

[jrev005]

^^Immunostain with a biotin conjugated monoclonal antibody targeted to an extracellular epitope. Detection with streptavidin tagged with the fluorescent molecule, phycoeryhthrin, and fluorescence was measured using a flow cytometer.

Im guessing, since Babcock_Hall mentioned that the two cysteine residues form an oxidised S-S bridge and hence are not very nucleophilic, then I shouldnt worry about my a,b-unsaturated carbonyl-containing test compounds causing a michael addition with these disulfide group. cells were only incubated for 2h at room temp.

[Babcock_Hall]

Do your proteins have other cysteine residues that are or might be reduced?