Studies on Interaction of Buffalo Brain Cystatin with Donepezil: An Alzheimer's Drug

Abstract

When drugs bind to a protein, the intramolecular structures can be altered, resulting in conformational change of the protein. Donepezil, an Acetyl Cholinesterase inhibitor (AChE), is commonly prescribed to patients with Alzheimer's disease (AD) to enhance cholinergic neurotransmission. It is the “first-line” agents in the treatment of Alzheimer's disease used to improve cognitive function in the disease. In the present study, a cysteine protease inhibitor (cystatin) has been isolated from buffalo brain using alkaline treatment, 40 to 60% ammonium sulphate fractionation and gel filtration chromatography on Sephadex G-75 with % yield of 64.13 and fold purification of 384.7. The purified inhibitor (Buffalo Brain Cystatin, (BBC)) was eluted as a single papain inhibitory peak which migrated as single band on native PAGE; however, on SDS-PAGE with and without beta mercaptoethanol (βME) BBC gave two bands of M W 31.6 and 12.4 KDa, respectively. The molecular weight determined by gel filtration came out to be 43.6 KDa. The UV spectra of cystatin on interaction with donepezil suggested a conformational change in the protein. The fluorescence spectra of BC-donepezil composite show structural changes indicating 40 nm red shift with significant increase in fluorescence intensity of cystatin in the presence of donepezil representing an unfolding of cystatin on interaction, which is an indication of side effect of donepezil during the use of this drug.

Interaction of almond cystatin with pesticides: Structural and functional analysis

“…On increasing Cu(II) hydroxide indicating that the binding perturbs the microenvironment of the aromatic amino acid residues, leading to the unfolding of the protein. 43 As shown in Figure 5, maximum unfolding was observed at 5μM concentration of the Cu(II) hydroxide.…”

“…These observations are concomitant with the UV absorption spectra. On increasing Cu(II) hydroxide concentration, the binding of almond cystatin with Cu(II) hydroxide led to an increase in the fluorescence intensity of the almond cystatin, indicating that the binding perturbs the microenvironment of the aromatic amino acid residues, leading to the unfolding of the protein . As shown in Figure , maximum unfolding was observed at 5μM concentration of the Cu(II) hydroxide.…”

“…So as to determine changes in the cystatin structure upon pesticide interaction, UV absorption spectra were recorded for the almond cystatin incubated with varying concentrations of the said pesticides (Figure A‐C). In the case of pendimethalin, the intensity of absorption of the samples increases with increasing concentrations of pendimethalin (1μM‐5μM); this slight increase in absorption intensity may be due to disruption or perturbation of absorbing groups (Figure A) …”