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If it is a heme or ironsulfur cluster then the reduced and oxidized forms have distinctly different spectra. Take a spectrum of your protein reduced with a few grains of dithionite and oxidized with a trace of hydrogen peroxide or ferricyanide (take another spectrum of ferricyanide alone since it absorbs at ~421 nm) eab On 11/26/2014 11:57 AM, Guenter Fritz wrote: > Hi Monica, > > what is the reducing agent the protein gets the electron from? > Or is it simply unspecific oxidation of protein side chains? > > Do you know what are the coordinating residues of Fe3+ / Fe2+ ? > > Dependent on the coordination Fe3+ has some (often very weak) d-d > transitions (400-500 nm) which are absent in Fe2+. > > Best method of choice to check whether you have Fe3+ or Fe2+ is EPR. > > Godd luck! > Guenter > > >> >> I am working on a protein which binds to its ligand in a particular >> state i.e. Fe3+ state (active) and reduces it to Fe2+ state >> (inactive). I am trying to set up crystallisation trials with the >> ligand but seems like difficult to get them. However i just needed an >> advice here, is there any way to know whether the protein is in Fe2+ >> state or Fe3+ state or how can i make the protein to exist in more of >> Fe3+ state so that i increase the probability of ligand binding to the >> protein. >> >> Your advice is highly appreciated. >> Thanks in advance !! >> >> Cheers >> Monica >