Our goal is to release the heme groups from myoglobin/hemoglobin prior to an investigation of the heme and its specific ligand. Acid is not an option since it could possible release the ligand we want to study.
Without knowing what you are trying to, it's really hard to make a suggestion, because the only methods I know (and this was many years ago) would not preserve a ligand:heme complex after release from the protein. It would seemingly be much easier to use other methods to detect the ligand while leaving the protein:heme intact in its native form.
Thank you for the comment Ted Carver.
We are looking at fraudulent addition og CO gas and nitrite in tuna fish, forming carboxymyoglobin and nitrosomyoglobin respectively, which make the tuna look fresh because of the red color. We have used spectroscopy to distinguich the two, but the UV spectra become complicated and it would be great if we could release the heme-ligands and separate them through chromatography.
Hmm, okay - disclaimer -I know a lot about myoglobin and its ligands, not so much about meat colorant testing - if the absorption spectra of the mixtures of native protein are complicated, there are couple of other tricks you can use instead, depending on why its complicated: 1.) you can add an oxygen scavenger/reductant, which will convert any oxyglobin to deoxy eventually; 2) you can use ion exchange chromatography to resolve Fe(II) and Fe (III) globin species as well as different globins. Also, if you let the samples oxidize for a period of time, then what is left should be CO, NO bound to Fe(II) plus the oxidized Fe(III) globins. So there are several ways to resolve the spectra and keep the CO, NO, without heme extraction.
Keeping the heme reduced as Fe(II) after extraction, with all ligands bound to the pentacoordinate heme, is a challenge to be avoided I would think.
Thank you very much for the answer. I will consider your ideas and discuss next move with my colleagues.
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