Drug protein interactions have gained considerable attention over the past many years. In the current communication the association of muscle cystatin (MC) with anti-rheumatic drugs methotrexate and dexamethasone was studied by thiol proteinase inhibitor assay, ultra violet (UV) absorption, fluorescence spectroscopy, and fluorescence transform infra-red spectroscopy (FTIR). A static pattern of quenching was noticed between muscle cystatin and methotrexate (MTX). Binding constant (Ka) of methotrexate to muscle cystatin was found to be 1 × 10-7 M-1 and the stoichiometry of binding was calculated to be one. Fluorescence measurement of the emission quenching reveals that the quenching process of cystatin by dexamethasone (DXN) was also static. The stoichiometry of binding and binding constant was also obtained. Additional evidence regarding MTX-MC and DXN-MC was obtained from UV spectroscopy and FTIR spectroscopic results. Such spectroscopic studies would help in modelling new candidate drugs for rheumatoid arthritis based on their cystatin binding profile.Communicated by Ramaswamy H. Sarma.

Cysteine proteinase inhibitors play an essential role in maintaining the proper functioning of all living cells by virtue of its thiol protease regulatory properties. Chemical denaturation of a new variant of cystatin super family has been studied by various biophysical techniques in order to characterize the unfolded and denatured state. Denaturation of garlic phytocystatin (GPC) has been investigated using urea and guanidine hydrochloride (GdnHCl). Different biophysical techniques such as intrinsic fluorescence, circular dichroism and FTIR exhibited an altered structure of garlic phytocystatin with increasing concentration of denaturant. The inhibitory activity of GPC decreases with increasing concentration of denaturant. Increased fluorescence intensity along with red shift reflects the unfolding of GPC at higher concentration of denaturant. GdnHCl induced unfolding showed presence of indiscernible intermediate as followed by ANS binding studies. However, denaturation by urea did not show any intermediates. Mid-point transition was observed at 4.7±0.1M urea and 2.32±0.1M GdnHCl. Circular dichroism and FTIR results indicate the 50% loss of secondary structure at 5M urea and 2.5M GdnHCl. This study provides intriguing insight into the possible alteration of structure, stability and function of GPC induced by urea and GdnHCl.