**3. Methods**

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%.

**65**

*Putative Involvement of Thiol Protease Inhibitor in the Function of Alzheimer Drug*

Papain inhibitory fractions of peak-I were pooled, concentrated, and checked for purity. Five milliliter fractions were collected and assayed for protein and cystatin activity. Homogeneity of the preparation was investigated by 7.5% PAGE [20].

Brain cystatin (BC) (1 M) was incubated for 30 min with subsequent higher concentration of donepezil in 0.05 M sodium phosphate buffer pH 7.5 in a final reaction volume of 1 mL at room temperature. The same buffer is used for preparation of drug solutions. Fluorescence measurements were carried out on a Shimadzu Spectroflourimeter model RF-5301PC (Shimadzu, Japan) equipped with a 150 W Xenon lamp and a slit width of 10 nm at 298 K. The fluorescence was recorded in wavelength region 300–400 nm after exciting the protein at 280 nm. The slits were set at 10 nm for excitation and emission. The path length of the sample was 1 cm.

The UV measurement of brain cystatin in the presence and absence of drug was made in the range of 200–300 nm, and the inhibitor (cystatin) concentration was fixed at 1 M, while the drug concentration was varied from 0.16 to 1.6 M. Absorption spectra were recorded on a double-beam Shimadzu UV-vis spectropho-

The inhibitory activity of the purified inhibitor (BC) under native conditions was assessed by its ability to inhibit caseinolytic activity of papain by the method of Kunitz [21]. The inhibitor (1 M) was incubated with increasing concentrations of donepezil at 25°C for 30 min before the activity was measured. Activity of

Alzheimer's disease is a progressive brain disorder that gradually destroys a person's memory and ability to learn, reason, and make judgments. AChE is responsible for degradation of the neurotransmitter acetylcholine (ACh) in the synaptic cleft of neuromuscular junctions and of neuronal contacts in the central nervous system [22, 23]. Donepezil belongs to the important class of acetylcholinesterase inhibitors (AChEIs) [24]. The results of the interaction of donepezil with cystatin

**5.2 Intrinsic fluorescence studies of cystatin in the presence of donepezil**

Cystatin (1 M) was incubated with various concentrations of donepezil varying from 2 to 10 M for 30 min. The fluorescence was recorded in the wavelength region of 300–400 nm after exciting the protein solution at 280 nm for total protein fluorescence. Donepezil caused unfolding of the cystatin as indicated by

**4.3 Activity measurement of brain cystatin in the presence of donepezil**

*DOI: http://dx.doi.org/10.5772/intechopen.83578*

**4.1 Fluorescence spectra of brain cystatin with drug**

**4.2 UV spectra of cystatin in the presence of donepezil**

tometer UV-1700 using a cuvette of 1 cm path length.

**5.1 Interaction of donepezil with brain cystatin**

untreated BC was taken as 100%.

**5. Results**

are given below.

**4. Spectroscopic studies**

Papain inhibitory fractions of peak-I were pooled, concentrated, and checked for purity. Five milliliter fractions were collected and assayed for protein and cystatin activity. Homogeneity of the preparation was investigated by 7.5% PAGE [20].
