Characterization of doxorubicin binding site and drug induced alteration in the functionally important structural state of oxyhemoglobin.
Doxorubicin (DOX) binding to hemoglobin (Hb) was studied to investigate the drug induced protein dysfunction. The features of anti-tumor drug doxorubicin infused structural perturbation of human Hb were studied by circular dichroism (CD). The mechanism of DOX-Hb binding was elucidated by steady-state and synchronous fluorescence spectroscopy. The Stern-Volmer analysis indicated that the binding of Hb to DOX is characterized by more than one high affinity binding site with the association constants of the order of 10(5). Hydrophobic probe ANS was employed to elucidate the drug binding site. Binding mode expounded by thermodynamic parameters implied the role of hydrogen bonding, electrostatic and hydrophobic interaction in stabilizing the complex. The molecular distance between donor (Hb) and acceptor (DOX) was calculated according to Förster's theory of energy transfer. Fourier transform infrared (FT-IR) spectroscopy provides an insight to the changes occurring in protein on DOX binding. Treatment of Hb with DOX resulted in a dose dependent fragmentation of protein. The quantitative analysis revealed the release of acid soluble amino groups from the photoexcited Hb-DOX mixture. The free radical mediated degradation was suggested by its rescue on mannitol and superoxide dismutase (SOD) appliance. The loss of protein band further corroborates the concentration dependent Hb fragmentation. The molecular modeling complies with the thermodynamic data of forces involved in DOX binding and depicts its interaction in the proximity of oxygen binding pocket of Hb. Thus, this study enriches our understanding of the interaction dynamics of anticancer drugs to the physiologically important protein Hb.
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh-202002, India.
Shahper N
SN
Khan
Barira
B
Islam
Ragothaman
R
Yennamalli
Qamar
Q
Zia
Naidu
N
Subbarao
Asad U
AU
Khan
D000903:Antibiotics, Antineoplastic / Q000737:chemistry; D001665:Binding Sites; D002021:Buffers; D002942:Circular Dichroism / Q000379:methods; D004317:Doxorubicin / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D015394:Molecular Structure; D010108:Oxyhemoglobins / Q000378:metabolism*; D010710:Phosphates / Q000737:chemistry; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods; D017550:Spectroscopy, Fourier Transform Infrared; D055672:Static Electricity; D013816:Thermodynamics
D000903:Antibiotics, Antineoplastic; D002021:Buffers; D010108:Oxyhemoglobins; D010710:Phosphates; D004317:Doxorubicin