Interaction Of Methemoglobin With Methotrexate Essay

2275 words - 9 pages

Hemoglobin is a metalloprotein that conveys oxygen [1] 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 [2] 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 [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 AuNPs distinguished by cytotoxic effects on human choriocarcinoma cells [12] and MTX liposome were used as oral drug delivery carriers [13].
A LC-MS/MS based method was recently used to estimate MTX polyglutamate concentration in the red blood cell [14]. Recent spectroscopic studies reveal 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. Several studies reveal the interaction of flavonoids [17][18][19], dyes [20][21][22], allosteric effectors [23] or other organic compounds [24][25] with hemoglobin.
Moreover, some studies focused on interaction of various antibiotics as oxytetracycline [26], chloramphenicol [27] and cephalosporines [28] with hemoglobin:
In particularly, hemoglobin interacts with several drugs: docetaxel [29], alprazolam-a sedative and antidepressant drug [30], tramodol-a synthetic analgesic [31]. However, few studies were focused on anticancer drugs interaction with hemoglobin. Thus, 5-azacytidine interacts with both HSA and hemoglobin [31].
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 spectra
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).

Fluorescence measurements
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...

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