Fluorescence Studies on the Binding Affinity and Determination of Vitamin B12 in the Presence of Fibrinogen.

The binding properties between vitamin B12 (vitB12, cyanocobalamin) and fibrinogen (Fib) were investigated by UV-vis absorption and steady-state/three-dimentional (3D) fluorescence spectra techniques as well as molecular docking. The experimental results showed that the intrinsic fluorescence of Fib quenched by vitB12 with static mechanism to form a non-fluorescent complex. The positive signs of thermodynamic parameters, ΔH (92.18 kJ/mol) and ΔS (433.5 J/molK), indicated that the hydrophobic forces were dominant in the binding mode. The molecular docking data were found to be in agreement with these experimental results and were confirmed by three hydrophobic interactions between the Trp430, Try390 residues of Fib and the vitamin. 3D spectra showed that fibrinogen undergoes a conformation change when it interacts with vitB12. Based on non-radiative energy transfer theory, binding distance was calculated to be 3.94 nm between donor (tryptophan residues of Fib) and acceptor (vitB12). The limit of detection (LOD) of vitB12 was calculated as 2.08 µM in the presence of fibrinogen. The relative standard deviation (RSD) of method was 4.28% for determinations (n = 7) of a vitB12 solution with the concentration of 7.80 µM.