Effect of temperature on the methotrexate – BSA interaction: Spectroscopic study

Rheumatoid arthritis (RA) is an autoimmune and chronic inflammatory illness which affects about one percent of the world’s population.Methotrexate (4-amino-10-methylfolic acid) (MTX) also known as amethopterin is commonly used to treat rheumatoid arthritis (RA). It is transported in the circulary system as a complex with serum albumin.The aim of this study was to investigate the interactions of MTX with transporting protein with the use of spectroscopic methods.The binding of MTX to bovine serum albumin (BSA) was studied by monitoring the changes in the emission fluorescence spectra of protein in the presence of MTX at excitation wavelength of 280 nm and 295 nm. The quenching of protein fluorescence at temperature range from 298 K to 316 K was observed.Energy transfer between methotrexate and fluorophores contained in the serum albumin structure was found at the molar ratio MTX:BSA 7.5:1.The relative fluorescence intensity of BSA decreases with increase of temperature. Similar results were observed for BSA excited with 280 nm and 295 nm at the same temperature range. The presence of MTX seems to prevent these changes. Temperature dependence of the binding constant has been presented. The binding and quenching constants for equilibrium complex were calculated using Scatchard and Stern–Volmer method, respectively.The results show that MTX forms π–π complex with aromatic amino acid residues of BSA. The binding site for MTX on BSA was found to be situated in the hydrophobic IIA or IB subdomain where the Trps were located. The spontaneity of MTX-BSA complex formation in the temperature range 298–316 K was ascertained.