Illustration of fluorescence quenching mechanism of heme proteins using stern volmer quenching system with determination of positioning of tryptophan residues

Structural conformation of proteins can be determined using spectroscopic measurements. Fluorescence activity of proteins is one of the primary criterions for studying biophysical characteristics. Structural elucidation of proteins is important for drug developments.Fluorescence quenching of Cytochrome C, Myoglobin and Leghemoglobin were carried out using acrylamide and Potassium Iodide (KI) solutions at room temperature. The quenching activity occurs due to excited state reactions, energy transfer and complex formations. Stern Volmer plot and quenching constants were obtained using the quenching systems. Due to tryptophan residues buried in the interior, mostly native heme proteins did not show a linear Stern Volmer plot; however, when denatured by guanidine hydrochloride, exposed tryptophan residues resulted in linear Stern Volmer system. Before denaturation, the Stern Volmer plot showed a polynomial nature. Denaturation resulted in increased exposure of the fluorophore to the quencher, which further resulted in linear Stern Volmer curve. Acrylamide quenching showed similar results in Stern Volmer quenching models of the three heme proteins, where KSV varied from 1.475, 16.83 and 0.04 for Cytochrome c, Myoglobin and Leghemoglobin. Fluorescence quenching study provided insight into the protein’s micro environment, containing tryptophan residues. This kind of study assisted in identification of Tryptophan positioning of the heme proteins.