Abstract
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•Fluorescence quenching studies on the interaction between riboflavin (Rib) and aquacobalamin (H2OCbl).•The results suggest the static quenching of Rib by H2OCbl through a 1:1 complex formation between Rib and H2OCbl.•The results suggest the non-radiative Förster energy transfer (FRET) between Rib and H2OCbl.•The thermodynamic parameters indicate that the binding process is spontaneous.
The interaction between two members of the vitamin B group, vitamin B2 (Riboflavin, Rib) and vitamin B12 (aquacobalamin, H2OCbl), was investigated in an aqueous solution by using fluorescence quenching and UV–vis absorption spectroscopy. The effect of pH on the interaction between Rib and H2OCbl was studied in different buffer solutions (pH = 3.4, 7.4, and 9.0) and at different temperature conditions (293, 303, and 308 K). The results demonstrate that the fluorescence intensity of Rib is significantly quenched by H2OCbl at all pH conditions. The experimental data shows a linear correlation to the Stern-Volmer equation suggesting that one mechanism of quenching takes place. The values of Stern–Volmer fluorescence quenching constant (KSV) were found to be 1.8 × 104, 1.4 × 104, and 1.2 × 104 M−1 at pH 3.4, 7.4, and 9.0, respectively. The values of the quenching rate constant (kq) are higher than the rate constant limit for the dynamic quenching, inferring that the fluorescence quenching of Rib by H2OCbl takes place through a static quenching mechanism. The absorption and fluorescence data fit will to Benesi-Hildebrand equation for a 1:1 inclusion complex between Rib and H2OCbl. The driving force for the electron transfer between Rib and H2OCbl system (ΔGet = −65.0 KJ/mol) is negative indicating that the electron transfer from the excited Rib to H2OCbl is thermodynamically favored. The donor–acceptor distance was determined according to Förster resonance energy transfer theory (FRET), r = 4.0 nm < 8 nm, suggesting the probability of FRET between Rib and H2OCbl. The thermodynamic parameters were calculated to be ΔH⁰ = −21.3 KJ mol−1, ΔS⁰ = 6.6 J mol−1 K−1, indicating that the electrostatic interaction has an important role in the reaction between Rib and H2OCbl.