Steady-state fluorescence emission from the fluorescent probe, 5-iodoacetamidofluorescein, bound to hemoglobin.
In the past, fluorescence emission from an extrinsic fluorophore bound to heme-proteins would only be studied with the removal of the heme since fluorescence from the fluorophore could not be detected using right-angle optics. Using front-face fluorometry, a significant steady state emission signal originating from the probe bound to hemoglobin is detected. This is the first report of the detection of extrinsic fluorescence of a probe bound to a heme-protein. We also demonstrate that the extrinsic probe, 5-iodoacetamidofluorescein, is covalently bound to hemoglobin, specifically at beta 93 Cysteine. Ligand binding results in a change in the fluorophore fluorescence intensity as predicted by hemoglobin crystallographic studies. Efficiency of energy transfer measurements are made.
D003545:Cysteine / Q000378:metabolism; D004592:Electrophoresis, Starch Gel; D005452:Fluoresceins / Q000378:metabolism*; D005470:Fluorometry / Q000379:methods; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D008433:Mathematics; D013050:Spectrometry, Fluorescence
D005452:Fluoresceins; D006454:Hemoglobins; C015296:5-iodoacetamidofluorescein; D003545:Cysteine