Investigation on the interaction between tamoxifen and human holo-transferrin: determination of the binding mechanism by fluorescence quenching, resonance light scattering and circular dichroism methods.
The interaction between tamoxifen (TMX) and human serum transferrin (HTF) was for the first time studied at varying pH values by fluorescence spectroscopy, circular dichroism (CD) and resonance light scattering (RLS). The fluorescence spectroscopy experiments were performed in order to study conformational changes, possibly due to a discrete reorganization of tryptophan residues during TMX-HTF binding at varying ligand concentrations, as well as quenching properties of the drug-serum transferrin complex and the differentiation between static and dynamic quenching. The binding affinity and number of binding sites were obtained for the TMX-HTF interaction at different pH. Second derivative fluorescence spectroscopy was employed for monitoring the complex and characterizing the transitions taking place in the environments of tyrosine and tryptophan (mainly tryptophans) in proteins. The variation of the K(SV) value suggested that hydrophobic and electrostatic interactions were the predominant intermolecular forces stabilizing the complex. The RLS technique was utilized to determine the protein type, and to investigate the effect of anticancer drugs on its determination. This is the first report of its kind. An explanation is also given of the enhancement in RLS intensity, which attributed to the new complex formation between TMX and the protein a self-aggregation process and the formation of a precipitate HTF occurred and a micelle came into being when the amount of TMX was augmented. The great increase of polarizability was one of the important factors for the enhancement of RLS and the formation of complexes. The results from synchronous fluorescence spectroscopy showed that the micro-environment around tryptophan and tyrosine demonstrated a faint red shift. The circular dichroism data revealed that the presence of TMX decreased the α-helix content of HTF and induced the remarkable unfolding of the polypeptides of the protein. This confirmed certain micro-environmental and conformational changes of the HTF molecule. The binding distance (r) between TMX and the tryptophan residue of HTF was obtained according to the Förster theory of non-radioactive energy transfer. This study on the interaction of drugs with HTF should prove helpful for realizing the distribution and transportation of drugs in vivo, elucidating the action mechanism and dynamics of a drug at the molecular level. It should moreover be of great use for understanding the pharmacokinetic and pharmacodynamic mechanisms of the drug.
Sarzehi
Sareh
S
Department of Biology, Faculty of Sciences, Islamic Azad University-Mashhad Branch, Rahnamaee Avenue, Mashhad, Iran.
Chamani
Jamshidkhan
J
D002942:Circular Dichroism / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008027:Light; D017711:Nonlinear Dynamics; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012542:Scattering, Radiation; D013056:Spectrophotometry, Ultraviolet; D013629:Tamoxifen / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics; D017186:Titrimetry; D014168:Transferrin / Q000737:chemistry / Q000378:metabolism*
D014168:Transferrin; D013629:Tamoxifen