**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

enhancement in fluorescence intensity accompanied by the red shift of 40 nm as compared to –max of native cystatin (340 nm), while the drug (native) shows –max at 370 nm. However at 1.6 M, when it forms complex with cystatin, there was a shift in –max of 10 nm with significant enhancement in fluorescence intensity (**Figure 4**).

Cystatin (1 M) was incubated with various concentrations of donepezil varying from 0.16 to 1.6 M for 30 min. The fluorescence was taken in the range of wavelength 300–400 nm after exciting the protein solution at 280 nm for total protein fluorescence. The slits were set at 10 nm for excitation and emission. The path length of the sample was 1 cm in the final reaction volume of 1 mL in 0.05 M sodium phosphate buffer pH 7.5.

#### **5.3 UV-vis spectra of cystatin in the presence and absence of donepezil**

Cystatin concentrations were fixed at 1 M, while the donepezil concentrations varied from 0.16 to 1.6 M. Absorption spectra of native cystatin and in the presence and absence of donepezil were recorded in the range of 200–300 nm. The UV absorption intensity of cystatin increased with increasing concentration of donepezil concentration; however, the slight decrease in absorption intensity may be due to disruption or perturbation of absorbing groups (**Figure 5**).

#### **5.4 Inhibitory activity of cystatin in the presence of donepezil**

A change in the inhibitory activity of cystatin with increasing concentration of donepezil is shown in **Table 1**. The effect of donepezil on cystatin function was assessed by monitoring its changes in antiproteolytic activity by caseinolytic assay of papain [21]; 1 M of cystatin was incubated with increasing concentration of

#### **Figure 4.**

*Fluorescence spectra of cystatin in the presence and absence of donepezil. Cystatin (1 μM) was incubated with various concentration of Donepezil varying from 0.16 to 1.6 μM for 30 min. The fluorescence was recorded in the wavelength region 300–400 nm after exciting the protein solution at 280 nm for total protein fluorescence. The slits were set at 10 nm for excitation and emission. The path length of the sample was 1 cm in the final reaction volume of 1 ml in 0.05 M sodium phosphate buffer pH 7.5.*

**67**

**Table 1.**

*Inhibitory activity of cystatin in the presence of donepezil.*

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

donepezil (0.16–1.6 M). Exposure of cystatin to increasing concentration of donepezil resulted in rapid decline of antiproteolytic activity; 85% decline in the activity was seen at 1.6 M of donepezil with more than half of the inactivation of cystatin

*UV-vis spectra of cystatin in the presence and absence of donepezil. Cystatin concentrations were fixed at 1 μM, while the donepezil concentration was varied from 0.16 to 1.6 μM. Absorption spectra of native cystatin in the presence and absence of donepezil were recorded in the range of 200–300 nm cuvette of 1 cm path length for* 

**Table 1** shows changes in the inhibitory activity of brain cystatin after its incubation for its inhibitory activity with increasing concentrations of donepezil. Cystatin (1 M) was treated with varying concentrations of donepezil (0.16–1.6 M) for 30 min in the final reaction volume of 1 mL in 0.05 M sodium phosphate buffer pH 7.5.

Purity of brain cystatin was confirmed by 2-D gel electrophoresis; purified cystatin was run in one dimension on the isoelectric focusing, and after this, it was run in second dimension on 12.5% PAGE. On 2-D gel electrophoresis, BC migrated as a single

**S.no Drug concentration % Remaining inhibitory activity of cystatin**

*All data are expressed as mean ± SE for three different sets of experiments; statistical significance was conducted employing one-way ANOVA. A probability level of 0.05 was selected showing that results are significant.*

*The table shows changes in the inhibitory activity of brain cystatin after its incubation for its inhibitory activity with increasing concentrations of donepezil. Cystatin (1 μM) is treated with varying concentrations of donepezil (0.16–1.6 μM) for 30 min in the final reaction volume of 1 ml in 0.05 M sodium phosphate buffer pH 7.5.*

 Cystatin alone 100 Cystatin + 0.16 μM donepezil 57 ± 0.623 Cystatin + 0.32 μM donepezil 40 ± 0.938 Cystatin + 0.64 μM donepezil 38 ± 0.772 Cystatin + 0.96 μM donepezil 24 ± 0.932 Cystatin + 1.6 μM donepezil 15 ± 0.680

which was taking place at concentration as low as 0.32 M.

*30 min in the final reaction volume of 1 ml in 0.05 M sodium phosphate buffer pH 7.5.*

**5.5 2-D gel electrophoresis**

**Figure 5.**

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

*Putative Involvement of Thiol Protease Inhibitor in the Function of Alzheimer Drug DOI: http://dx.doi.org/10.5772/intechopen.83578*

#### **Figure 5.**

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

intensity (**Figure 4**).

sodium phosphate buffer pH 7.5.

enhancement in fluorescence intensity accompanied by the red shift of 40 nm as compared to –max of native cystatin (340 nm), while the drug (native) shows –max at 370 nm. However at 1.6 M, when it forms complex with cystatin, there was a shift in –max of 10 nm with significant enhancement in fluorescence

Cystatin (1 M) was incubated with various concentrations of donepezil varying from 0.16 to 1.6 M for 30 min. The fluorescence was taken in the range of wavelength 300–400 nm after exciting the protein solution at 280 nm for total protein fluorescence. The slits were set at 10 nm for excitation and emission. The path length of the sample was 1 cm in the final reaction volume of 1 mL in 0.05 M

**5.3 UV-vis spectra of cystatin in the presence and absence of donepezil**

disruption or perturbation of absorbing groups (**Figure 5**).

**5.4 Inhibitory activity of cystatin in the presence of donepezil**

Cystatin concentrations were fixed at 1 M, while the donepezil concentrations varied from 0.16 to 1.6 M. Absorption spectra of native cystatin and in the presence and absence of donepezil were recorded in the range of 200–300 nm. The UV absorption intensity of cystatin increased with increasing concentration of donepezil concentration; however, the slight decrease in absorption intensity may be due to

A change in the inhibitory activity of cystatin with increasing concentration of donepezil is shown in **Table 1**. The effect of donepezil on cystatin function was assessed by monitoring its changes in antiproteolytic activity by caseinolytic assay of papain [21]; 1 M of cystatin was incubated with increasing concentration of

*Fluorescence spectra of cystatin in the presence and absence of donepezil. Cystatin (1 μM) was incubated with various concentration of Donepezil varying from 0.16 to 1.6 μM for 30 min. The fluorescence was recorded in the wavelength region 300–400 nm after exciting the protein solution at 280 nm for total protein fluorescence. The slits were set at 10 nm for excitation and emission. The path length of the sample was 1 cm in the final reaction* 

**66**

**Figure 4.**

*volume of 1 ml in 0.05 M sodium phosphate buffer pH 7.5.*

*UV-vis spectra of cystatin in the presence and absence of donepezil. Cystatin concentrations were fixed at 1 μM, while the donepezil concentration was varied from 0.16 to 1.6 μM. Absorption spectra of native cystatin in the presence and absence of donepezil were recorded in the range of 200–300 nm cuvette of 1 cm path length for 30 min in the final reaction volume of 1 ml in 0.05 M sodium phosphate buffer pH 7.5.*

donepezil (0.16–1.6 M). Exposure of cystatin to increasing concentration of donepezil resulted in rapid decline of antiproteolytic activity; 85% decline in the activity was seen at 1.6 M of donepezil with more than half of the inactivation of cystatin which was taking place at concentration as low as 0.32 M.

**Table 1** shows changes in the inhibitory activity of brain cystatin after its incubation for its inhibitory activity with increasing concentrations of donepezil. Cystatin (1 M) was treated with varying concentrations of donepezil (0.16–1.6 M) for 30 min in the final reaction volume of 1 mL in 0.05 M sodium phosphate buffer pH 7.5.

## **5.5 2-D gel electrophoresis**


Purity of brain cystatin was confirmed by 2-D gel electrophoresis; purified cystatin was run in one dimension on the isoelectric focusing, and after this, it was run in second dimension on 12.5% PAGE. On 2-D gel electrophoresis, BC migrated as a single

*All data are expressed as mean ± SE for three different sets of experiments; statistical significance was conducted employing one-way ANOVA. A probability level of 0.05 was selected showing that results are significant. The table shows changes in the inhibitory activity of brain cystatin after its incubation for its inhibitory activity with increasing concentrations of donepezil. Cystatin (1 μM) is treated with varying concentrations of donepezil (0.16–1.6 μM) for 30 min in the final reaction volume of 1 ml in 0.05 M sodium phosphate buffer pH 7.5.*

#### **Table 1.**

*Inhibitory activity of cystatin in the presence of donepezil.*

#### **Figure 6.**

*2-D gel electrophoresis of purified BC: after isoelectric focusing, IPG was run horizontally over 12.5% gel between the glass slabs. A single dot was obtained toward the negative side of the IPG strip.*

dot further supporting the purity nature of BC. The position of the spot was approximately in the range of 7–8 pH on IPF strip (pH 3–10) suggesting the pl of BC as 7–8.

All data are expressed as mean ± SE for three different sets of experiments; statistical significance was conducted employing one-way ANOVA. A probability level of 0.05 was selected showing that results are significant (**Figure 6**).

### **6. Discussion**

The drug inducing changes in protein function leading to adverse side effects is the area of continual scientific investigation [25, 26].

Even small structural differences in protein conformation can lead to drastic changes in functional parameters [26]. Addition of small molecules such as many drugs, particularly those with local anesthetics, tranquilizers, and antidepressants, can bind to the native state and can alter the delicate balance of various interactions in proteins [27–30].

The adverse drug reactions are triggered due to gathering of drug molecules at localized sites in the body causing their elevated concentration [31] and ligand-induced protein structure conformational changes [32]. The drugs used as medical therapy are unable to act in the specified area because of these intricate mechanisms; therefore, all the varied external parameters are taken into account which combine the study of the conformational modifications in proteins along with drugs resulting complexes causing any side effect which are paramount for the study. These studies enable us to understand how ligand affinity can be planned and how the protein conformation upon complexation can be managed [26] which is decisive in a massive range of imperative biochemical phenomena.

In the present work and structural and functional analyses of cystatin, a protein ubiquitously present in mammalian cells and tissues was studied which showed a significant increase in fluorescence intensity due to unfolding of cystatin in the presence of donepezil (**Figure 4**). Such kind of changes have also been documented earlier, after interaction of ligands (phytohormones, cytokinins, abscisic and gibberellic acids) with wheat germ agglutinin resulting in 60% increase in fluorescence intensity of native protein [33].

**69**

provided the original work is properly cited.

and Bilqees Bano2

\*

\*Address all correspondence to: bilqeesbano691@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 Department of Zoology, Faculty of Life Sciences, Aligarh Muslim University,

2 Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University,

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

Donepezil-cystatin complexation showed 40 nm red shift in λmax indicating exposure of aromatic residues to the solvent caused by conformational changes in the protein [34, 35]. Absorption spectral measurements of cystatin in the presence of drug showed a peak noticeable at 275 and 210 nm in spectra obtained at 0.16 M donepezil concentration (**Figure 5**). Suggesting changes mainly due to tryptophan

Thus, the results indicate that the UV absorption and fluorescence emission changes in donepezil-mediated interaction are due to conformational changes in cystatin mainly arising from interaction affecting the chromophoric groups of the protein which produce significant effect on the activity of cystatin. The study shows that in the presence of donepezil, cystatin gets unfolded which is a side effect of

There is a gradual decline in the cystatin activity with increasing drug concentration resulting in 62% loss at 0.64 M donepezil (**Table 1**). Further magnitude of decline was relatively smaller with increasing drug concentration up to 1.6

The knowledge about the pharmacokinetics and pharmacodynamics of the drug-protein interaction continues to expand. The increased information available to clinicians might help in optimizing the use of these agents in the management of patients with Alzheimer's disease and other diseases. The clinical utility of measur-

M. Cystatin retained only 15% of its antiproteolytic potential.

ing these parameters in daily practice awaits further research [37].

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

and tyrosine residues [36].

donepezil.

**Author details**

Aligarh, Uttar Pradesh, India

Aligarh, Uttar Pradesh, India

Fakhra Amin1

#### *Putative Involvement of Thiol Protease Inhibitor in the Function of Alzheimer Drug DOI: http://dx.doi.org/10.5772/intechopen.83578*

Donepezil-cystatin complexation showed 40 nm red shift in λmax indicating exposure of aromatic residues to the solvent caused by conformational changes in the protein [34, 35]. Absorption spectral measurements of cystatin in the presence of drug showed a peak noticeable at 275 and 210 nm in spectra obtained at 0.16 M donepezil concentration (**Figure 5**). Suggesting changes mainly due to tryptophan and tyrosine residues [36].

Thus, the results indicate that the UV absorption and fluorescence emission changes in donepezil-mediated interaction are due to conformational changes in cystatin mainly arising from interaction affecting the chromophoric groups of the protein which produce significant effect on the activity of cystatin. The study shows that in the presence of donepezil, cystatin gets unfolded which is a side effect of donepezil.

There is a gradual decline in the cystatin activity with increasing drug concentration resulting in 62% loss at 0.64 M donepezil (**Table 1**). Further magnitude of decline was relatively smaller with increasing drug concentration up to 1.6 M. Cystatin retained only 15% of its antiproteolytic potential.

The knowledge about the pharmacokinetics and pharmacodynamics of the drug-protein interaction continues to expand. The increased information available to clinicians might help in optimizing the use of these agents in the management of patients with Alzheimer's disease and other diseases. The clinical utility of measuring these parameters in daily practice awaits further research [37].
