**4. Spectroscopic studies**

*Redirecting Alzheimer Strategy - Tracing Memory Loss to Self Pathology*

inhibitor cystatins [1].

concentration in plasma [17].

cystatin effecting its activity?

**2. Materials**

used throughout.

**3. Methods**

Cystatins tightly bind and impede the activity of cathepsins; if the activity of cathepsins is not regulated, it instigates chronic diseases [13]. Senile plaque, cerebrovascular amyloid deposits, and neurofibrillary tangles in Alzheimer's disease are all the ramifications of imbalance between proteinases and their endogenous

Normal functioning of the brain is balanced by maintaining the level of acetylcholine and acetylcholinesterase inhibitor [16]. A previous report showed that donepezil binds along with HSA modifying it conformationally by effecting its free

The supplementation of donepezil was explored to find out any effect on cystatin (major regulator of thiol proteases: cathepsins B, H, and L, etc.) in the mammalian system. If the activity of these proteases is not controlled, it will lead to protease and antiprotease imbalance and repercussion to several diseases [18]. Therefore, it was thought worthwhile to investigate the donepezil cystatin binding and its role in the proper accomplishment of drug delivery and if it lead to kind of side effects as well as to gain knowledge about any conformational change in

The study shows that the imbalance of protease-antiprotease purportedly leads the way to Alzheimer's disease, while the presence of drug donepezil unfolds cystatin which becomes unfit to bind cathepsins leading to a number of diseases as a considerable side effect of the drug. As cystatins play significant role in several diseases like, cancer and cardiovascular diseases [19]. Therefore, the usage of

Papain (99% purity) was obtained from Sigma Chemical Company (St. Louis, USA). Donepezil (an Alzheimer drug) was purchased from Ranbaxy (India). The solutions were prepared in 50 mM phosphate buffer of pH 7.4. Salts were purchased from Merck (India). The protein concentration was determined spectrophotometrically. All other reagents were of analytical grade, and double distilled water was

Purification of brain cystatins. Buffalo brain whole mass (150 g) was brought fresh from slaughter house in a box containing ice packs. It was carefully washed and rinsed with water, eliminating thin membrane and nerves by forceps, and the whole brain tissue was homogenized in 50 mM sodium phosphate buffer (300 mL) of pH 7.5 containing 0.15 M NaCl, 3 mM EDTA, and 2% n-butanol. After centrifugation at 11,000 rpm for 15 min at 40°C, residue was cast off, and the supernatant was further processed. The procedure involved a combination of alkaline treatment (pH 11.0), ammonium sulfate fractionation, and gel filtration chromatography. The brain was homogenized and fractionated with ammonium sulfate between 40 and 60% saturation; the precipitated protein was then dialyzed against 50 mM sodium phosphate buffer pH 7.4 containing 0.1 M NaCl. Elution profile showed two protein peaks—one major and one minor called as peak-I and peak-II. Peak-I is conforming to high-molecular-weight buffalo brain cystatin with significant inhibitory activity and protein content; however, peak-II with insignificant protein concentration and low inhibitory activity was not taken into consideration for further studies. Peak-I named as BC was purified with fold purification of 384.72 and yield of 64.13%.

donepezil in such patients requires additional attention.

**64**
