Interaction of Human Hemoglobin with Methotrexate

Abstract

This study focuses on the interaction between methotrexate and human hemoglobin using steady-state ultravioletvisible and fl uorescence quenching methods. Fluorescence quenching was found to be valuable in assessing drug binding to hemoglobin. The quenching of methotrexate is slightly smaller than the quenching observed with related analogs (dihydrofolate and tetrahydrofolate). The quenching studies were performed at four different temperatures and various pH values. The number of binding sites for tryptophan is ~1. Parameter-dependent assays revealed that electrostatic forces play an essential role in the methotrexate-hemoglobin interaction. Furthermore, the complex was easily eluted using gel fi ltration chromatography.Keywords: hemoglobin, methotrexate, fl uorescence quenching, gel fi ltration. Introduction.Hemoglobin is a metalloprotein that transports oxygen [1] from the lungs to peripheral tissues such as the muscles, liver, and intestines. Human adult hemoglobin is a tetrameric blood protein, consisting of two pairs of α and β polypeptide chains. The hemoglobin tetramer contains six Trp residues. The intrinsic fl uorescence of hemoglobin originates mainly from a Trp residue located close to the heme moiety. Methotrexate (MTX) is a well-known inhibitor of dihydrofolate reductase [2], an enzyme that inhibits the synthesis of new DNA, and has been widely used in the treatment of various cancers. Since 1985, MTX has been used as an effective drug for the treatment of rheumatoid arthritis [3][4][5], psoriasis [6, 7], acute lymphoblastic leukemia [8], lymphoma, head and neck cancer [9], breast cancer [10], and osteosarcoma [11]. Additionally, MTX-conjugated gold nanoparticles distinguished by cytotoxic effects on human choriocarcinoma cells [12] and liposome-entrapped MTX were successfully used as oral drug-delivery systems [13]. A liquid chromatography-tandem mass spectrometry-based method was recently used to estimate the MTXpolyglutamate concentration in red blood cells [14]. Recent spectroscopic studies have revealed the interaction of MTX with nucleic acids [15] or serum albumin [16]. Various spectroscopic methods have been used to study the interaction of small organic molecules with proteins. Some studies have shown the interaction of fl avonoids [17-19], dyes [20-22], allosteric effectors [23-25], or other organic compounds [26, 27] with hemoglobin. Moreover, some studies have focused on the interaction of various antibiotics, namely oxytetracycline [28], chloramphenicol [29], and cephalosporins [30], with hemoglobin.In particular, hemoglobin interacts with several drugs: docetaxel [31], alprazolam, a sedative and antidepressant drug [32], and tramadol, a synthetic analgesic [33]. However, there are only a few studies that have focused on the interaction of anticancer drugs with hemoglobin. For example, 5-azacytidine interacts with both human serum albumin and hemoglobin [33]. In this study, the binding affi nity of MTX towards hemoglobin was studied.Experimental. Chemicals. Hemoglobin (human), whale ske...