Toward oxygen binding curves of single respiratory proteins

Oxygen binding curves of single molecules promise to discriminate between different models describing cooperativity because load distributions are accessible. Individual tarantula hemocyanins could be detected by fluorescence correlation spectroscopy using intrinsic tryptophan fluorescence as sensor of bound oxygen. However, imaging of immobilized proteins was not possible due to fast photo-bleaching. It is shown that tetra-methyl-carboxy-rhodamine (TAMRA), commonly used as a fluorescence label in single-molecule spectroscopy, can also be applied to monitor bound oxygen. The dye's fluorescence is quenched due to F?rster energy transfer to the oxygenated active sites of hemocyanin.