The binding mechanism and affinity of the interaction between levothyroxine (LT4) and bovine serum albumin (BSA) were investigated, both in solution using UV-Vis, Fourier-transform infrared spectroscopy (FT-IR), fluorescence and fluorescence resonance energy transfer (FRET), as well as by Surface Plasmon Resonance (SPR) with BSA confined to a gold-coated chips. Quenching of BSA fluorescence by LT4 combined with UV-Vis spectroscopy shows a ground-state complex formation that may be accompanied by a nonradiative energy transfer process. FT-IR revealed the changes induced by LT4 in the secondary structure of BSA molecules, due to the partial unfolding of BSA native structure upon LT4 binding. Scatchard approach allowed the determination of the binding constant K b (5.12 x 10 6 M -1 ) and the equilibrium dissociation constant K d (19.5 x 10 -6 M) which suggest a moderate binding, as well as the thermodynamic parameters, which correspond to an enthalpic process (-51.99 kJ mol -1 ), driven mainly by hydrogen bonds and van der Waals forces. Using SPR, first, the confinement of BSA onto the chip gold surface was optimized towards LT4 binding. Second, the binding affinity of LT4 with BSA was characterized using the Hill-Langmuir equation, which led to a moderate binding affinity (apparent K d = 67.6 x 10 -6 M). As preliminary results, both fluorescence quenching and SPR can be used as a stepping stone for the development of a spectroscopic biosensor for LT4 detection, with a limit of detection as low as 0.23 x 10 -6 M.
SIGNIFICANCEMost cellular and tissue functions are influenced by thyroid hormones, such as thyroxine (T4). Thus, these hormones influence the metabolism of various biomolecules and are related to a wide variety of medical conditions, of which, the best therapy is the hormone replacement using LT4 medicine. Therefore, the monitoring LT4 levels in the human body while undergoing LT4 treatment is of particular importance. Here we investigated the binding mechanism and affinity between LT4 and bovine serum albumin (BSA), one of the transporter proteins, by spectroscopic (UV-Vis, FTIR, fluorescence) and optical (SPR) methods. Although LT4-BSA binding was driven mainly by hydrogen and van der Waals bonds with moderate affinity, we found that fluorescence can be used for LT4 detection in nanomolar range.
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RTICLEFIGURE 1 (A) The UV spectrum of LT4 diluted in HEPES buffer solution, pH 7.4. (B) The absorbance of LT4 at pH in the range of 5.35 and 9.40, measured at 240 nm. (C) The representation of the 1 st derivative of the absorbance.FIGURE 2 (A)FT-IR curves of LT4 and BSA-LT4 between the 3,000 -2,800 cm −1 , corresponding to the NH2 stretching in LT4 is shifted from 2,954 cm-1 to 2,920 cm-1 in the BSA-LT4 complex; the peak from 2836 cm -1 in LT4 is completely attenuated in the BSA-LT4 complex; curve-fitted amide I band between the 1,700 -1,600 cm −1 for: (B) free BSA and (C) BSA-LT4. The amide I band is represented in black and the curves for the secondary structure elements are: blue for α helix, magenta ...