Intrinsic fluorescence determination of the interaction of luminol with hemoglobin

CHED 867

Randi E. Cattoi, rcattoi@daemen.edu, Derrick J. Swartz, dswartz@daemen.edu, and Matthew S. Ward, mward@daemen.edu. Department of Natural Sciences, Daemen College, 4380 Main Street, Amherst, NY 14226
The presumptive test for blood identification at a crime scene involves the interaction of iron(II) in the heme of hemoglobin with luminol. Data obtained from studies about how crime scene interferences and luminol interact with hemoglobin have produced inconclusive results. The binding of a ligand to a protein may alter the binding affinity of other sites within the molecule and potentially change the protein's conformation. A modification of the luminol-heme interaction may result from the conformational changes. The changes in the protein conformation based on shifts in fluorescence are monitored using intrinsic fluorescence spectrophotometry coupled with urea denaturation. Unfolding curves of fluorescence intensity as a function of urea concentration show a clear, 2-state, thermodynamically favored unfolding pattern with and without ligand.