**Abstract**

The intermolecular structure gets altered when drug-protein interaction takes place. It brings about alterations in the conformation of protein. An acetyl cholinesterase inhibitor (AChE) is the most used drug for patients who are suffering from Alzheimer's disease to curb its instigated symptoms. So, it is used as first-line defense in the insightful symptoms. This study is of concern with the interaction of cystatin purified from buffalo brain with its simple tri-step procedure including alkaline action, ammonium sulfate fractionation, and gel filtration chromatography on Sephadex G-75 with % yield of 64.13 and fold purification of 384.7. The inhibitor (brain cystatin (BC)) showed a single papain inhibitory peak drifted as single band on native PAGE; this purified inhibitor was interacted with donepezil to analyze the side effect of this drug since cystatin is an important regulatory protein that maintains the protease antiprotease balance. The conformational change was predicted when the UV spectra of cystatin was analyzed in the presence of donepezil contextual with the fluorescence spectra, but the fluorescence spectra showed 40 nm of red shift suggesting the change on interaction leading to a conclusion that donepezil is pertinent to imbalance to protease and antiprotease inhibitor perhaps the side effect of drug.

**Keywords:** acetyl cholinesterase (ach), acetyl cholinesterase inhibitor (AChE), Alzheimer's disease (AD), brain cystatin, (BC), thiol protease inhibitor
