We are purifying a copper protein that should have 4 moles of copper per mole of protein, but we don't know if the protein is fully loaded with copper. We don't have atomic absorption spectroscopy and how can we determine the total copper concentration in the protein? thanks for your help!
Difficult to orient you towards a specific determination method, without knowledge of the expected Copper concentration or the solubility of the Copper ions from your protein complex (easy to release or not ?).
Off the top of my head and without having to turn to mineralization followed by AA or ICP (which would be ideal) :
- all time classic is iodometric titration : reduction of Cu2+ by iodide I-, which produces iodine I2 that can be back titrated using thiosulfate S2O32-
- colorimetric determination by UV-Vis spectroscopy : a variety of methods do exist, using various ligands to produce a coloured complex from Cu2+
You will have to perform a 'wet' digestion procedure with Aqua Regia, neutalize then analyse colormetrically with a chelate. I think it was named 'Cuprion' (its been >40 years, so my memory is iffy sometimes).
You can destroy the protein, e.g. by incineration or with nitric acid. Dissolve the residue in nitric acid. Add excess ammonia water until a blue color develops and measure the absorbance, preferably not in the absorption maximum, but in the so-called isobestic point of the absorption spectrum, because the absorbances at the absorption maximum are too dependent on the ammonia concentration, and these concentrations may be slightly different from one solution to the other.
ZJ
Your going to have to do some calculations. I presume you can dry ash ~100 mg of protein and use the neocuproine method in APHA's (American Public Health Association) 'Standard Methods for the Examination of Water and Wastewater' 3-95, 1989 edition. The LOQ is listed at ~0.3 mg/25 mL.
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