Hemoglobin is a metalloprotein that conveys oxygen  from lungs to the peripheral tissues such as muscles, liver and intestines. Human adult hemoglobin (Hb) is a tetrameric blood protein, consisting of two pairs of and β polypeptide chains. The hemoglobin tetramer contains a total of 6 Tryptophanes. The intrinsic fluorescence of Hb originates mainly from a Trp residue located near to the heme moiety. Methotrexate, a well-known inhibitor of dihydrofolate reductase  an enzyme which inhibits the synthesis of new DNA, was widely used in the treatment of various cancers.
Since 1985, MTX has been used as an effective drug in rheumatoid arthritis , psoriasis , acute lymphoblastic leukemia  lymphoma, head and neck cancer , breast cancer  and osteosarcoma . Additionally, MTX-conjugated AuNPs distinguished by cytotoxic effects on human choriocarcinoma cells  and MTX liposome were used as oral drug delivery carriers .
A LC-MS/MS based method was recently used to estimate MTX polyglutamate concentration in the red blood cell . Recent spectroscopic studies reveal the interaction of MTX with nucleic acids  or serum albumin .
Various spectroscopic methods have been used to study the interaction of small organic molecules with proteins. Several studies reveal the interaction of flavonoids , dyes , allosteric effectors  or other organic compounds  with hemoglobin.
Moreover, some studies focused on interaction of various antibiotics as oxytetracycline , chloramphenicol  and cephalosporines  with hemoglobin:
In particularly, hemoglobin interacts with several drugs: docetaxel , alprazolam-a sedative and antidepressant drug , tramodol-a synthetic analgesic . However, few studies were focused on anticancer drugs interaction with hemoglobin. Thus, 5-azacytidine interacts with both HSA and hemoglobin .
The aim of this study was to estimate binding of MTX to hemoglobin.
Materials and methods
Hemoglobin (human) was purchased from Sigma (Germany), methotrexate solution (MTX, 100 mg/ml) from Ebewe (Austria) and Tris respectively HEPES from Roth (Germany).
UV–VIS absorption spectra were recorded using a Libra UV/Vis Spectrophotometer (Biochrom, UK) (with a Peltier thermostated cell holders maintained at 25 °C). The experiments were performed by adding microliter amounts of methotrexate to hemoglobin (µmolar range) dissolved in HEPES 50 mM buffer/NaCl 50 mM pH 7.4. For the UV and Vis-domains quartz cuvettes with a 1-cm path length were used (Helma, Müllheim, Germany).
All fluorescence spectra were recorded on a SFM-25 Spectrofluorimeter (Kontron Instruments S.P.A., Milan, Italy) equipped with 1.0 cm quartz cell and a thermostated bath. The appropriate blanks corresponding to the buffer or methotrexate were substracted in order to correct for background fluorescence. A 3.0 mL aliquot...