To analyse the change in the functionality of α₂M, antiproteinase assay was performed. The activity profile of protein was measured with increasing concentrations of 5-FU in the presence of light. To determine the protective effect of quercetin, it was preincubated with protein and then treated with 5-FU38. The reaction mixture consisted of 1 ml sample in 50 mM sodium phosphate buffer (pH 7.4) at room temperature (22 ± 1 ⁰C). After incubation for 1 hr, 0.161 nmol trypsin was added into the mixture and incubated for 15 min at 37ºC. After 15 min incubation, 0.45 nmol of soybean trypsin inhibitor (STI) was added into the reaction mixture and the incubation was continued for additional 15 min at 37ºC. Finally, 2.28 μmol of N-Benzoyl-DL-arginine-p-nitroanilide (BAPNA) was added and absorbance was observed at 410 nm.

The fluorescence spectra for native α₂M and 5-FU and quercetin were recorded on a Shimadzu RF-5301 spectrofluorometer (Tokyo, Japan) in a 10 mm path length quartz cell. The excitation wavelength was set at 280 nm and the emission spectra were recorded in the range of 300-400. The concentration of protein in the sample was 10 µM with varying concentration of 5-FU and the concentration of quercetin was 50 µM in the aliquot.

Synchronous fluorescence was recorded on a Shimadzu RF5301 spectrofluorophotometer, Japan. The wavelength range (250 – 400 nm) of fluorescence spectra selected for synchronous scanning was recorded at Δλ of 15 nm and 60 nm for tyrosine and tryptophan, respectively.

UV spectrum of native α₂M (10 µM) and α₂M incubated with quercetin (50 µM) with increasing concentration of 5-FU were recorded between 250 – 350 nm on a Shimadzu UV visible Spectrophotometer UV-1700 using a cuvette of 1 cm path length39.

FTIR spectroscopy was performed on an Interspec 2020 FTIR spectrometer. The samples were prepared by dissolving native α₂M (20 µM) and α₂M incubated with quercetin and 5-FU in 20 mM sodium phosphate buffer, pH 7.4. The spectra of native α₂M incubated with varying concentration of 5-FU and quercetin (50 µM) were analyzed. Amide I (1600 – 1700 cm^-1) is the region which is usually analyzed to confirm the change in the native structure of the protein. The scanning wave number was 1000 – 4000 cm^-140.

CD measurements were recorded on a JASCO-J815 spectropolarimeter equipped with a Peltier-temperature controller. Wavelength scan for native α₂M (20 µM) and quercetin with varying concentration of 5-FU were carried out. The temperature was maintained at 25°C and the speed was fixed at 100 nm/min with a response time of 1s. Each spectrum was the average of 3 scans and was recorded in the wavelength range of 200 – 250 nm using quartz cuvette of 0.1 cm path length.

The superoxide species (O₂^.-) generation was estimated by the nitro-blue-tetrazolium (NBT) assay41. A typical assay mixture was used containing 50 mM sodium phosphate buffer (pH 8.0), 300 µM NBT, 100 µM EDTA and 0.06% Triton-X-100 and α₂M (20 µM) treated with varying concentration of 5-FU (10 – 50 µM). The absorbance was recorded at 560 nm at different time intervals, against a blank which was without 5-FU.

Fluorescence microscopy was utilized to determine the formation of reactive oxygen species (ROS). Determination of ROS was performed using the dichlorofluorescein (DCF) method42. Protein was preincubated with quercetin for 1 hr, treated with 5-FU and incubated in the presence of white light. Then the sample was incubated with 10 μM dichloro-dihydro-fluorescein diacetate (DCFH-DA) for 1 hr at 37°C. The treated protein was placed on glass coverslips and visualized under a fluorescent microscope at 40 x magnification.

Results were shown as the mean and the standard deviation (SD values, with n=3 as the number of independent experiments.

Figure 1

Figure 2

The activity assay of native α₂M with varying concentration of 5-FU was measured in order to check the functional status of the protein. Firstly, we measured the antiproteinase activity of protein incubated with the drug in the presence of white light. Our results show that the activity of protein decreases with increasing concentration of 5-FU (10 – 50 µM). According to Figure 1a, the residual activity was found to be 87% at 10 µM 5-FU. When the concentration of the drug (50 µM) was increased, the remaining activity was found to be 48%. According to previous studies, 5-FU is known to generate free radicals in the presence of white light43. Therefore, the anti-proteolytic potential of the protein was compromised, which may be due to the formation of free radicals by the drug leading to changes in the functionality of the protein. We measured the protective effect of quercetin against the toxicity induced by 5-FU, since it is a flavonoid which is known to scavenge free radicals44. When the protein was preincubated with quercetin and then treated with 5-FU in the presence of light the resultant activity decreased, but not as much compared to 5-FU alone. Our results suggest that the residual inhibitory activity at 50 µM 5-FU was 59% when the protein was preincubated with quercetin (Figure 1b). Therefore, quercetin provides a protective role against 5-FU since it may scavenge free radicals that are generated by the drug.

Intrinsic fluorescence is mainly used to determine the conformational changes in the native form of the protein. The fluorescence spectra were recorded for the protein (20 µM) with increasing concentration of 5-FU (10 – 50 µM). The protein was excited at 280 nm and emission spectra were taken in the range 300 - 400 nm. The emission maximum (λmax/λem) for native α₂M was found to be 328 nm, as shown in Figure 2a. It is quite clear from the figure that with increasing concentration of 5-FU, the fluorescence intensity decreases continuously with a blue shift of 3 nm. The decrease in the intensity is due to the quenching in fluorescence due to the interaction between the protein and the drug. The quenching occurs mainly due to three intrinsic fluorophores in the protein i.e., phenylalanine, tyrosine and tryptophan45. The change in the fluorescence intensity can be correlated to the change in the structure of α₂M as the ligand binds to it46, which may also alter the function of the protein. Many in vitro studies suggest that quercetin can reduce the toxicity induced by anticancer drugs and provide a protective effect against free radical induced damage31. To evaluate the protective effect of quercetin against 5-FU-induced toxicity on α₂M, intrinsic fluorescence was recorded. The protein was first preincubated with quercetin and then treated with the drug in the presence of white light, intrinsic fluorescence was then recorded. As the results suggest, with varying concentrations of 5-FU the fluorescence intensity decreases, but when the protein was preincubated with quercetin, less quenching occurs (Figure 2b). So, we can conclude that in the presence of quercetin, α₂M regains its native structure which was damaged by the drug.

Synchronous fluorescence was performed to study the changes in the molecular environment in the vicinity of the chromophore. The spectral analysis provides information about the conformational changes around the tyrosine and tryptophan residues of the protein. The effect of fluorescence analysis of 5-FU on α₂M is shown in Figure 3. With the addition of drug, there was a decrease in the fluorescence intensity with a shifting of emissions towards a longer wavelength from 341 to 344 nm for tryptophan residues and towards a shorter wavelength from 309 to 305 nm for tyrosine residues. This decrease in the fluorescence intensity with varying concentration of 5-FU suggests changes in the surrounding microenvironment around tyrosine and tryptophan residues with a decrease in hydrophobicity around both residues47.

UV spectra was analyzed to determine the structural change in the native protein and to study the formation of a complex between protein and ligand48. It is a classical technique to determine the modification in the microenvironment of amino acid residues, particularly tryptophan, resulting in the change in the spectra or a shift in maximum absorbance/lambda max (λ_max)45. To examine the change in the native structure of α₂M with varying concentration of 5-FU, spectra was recorded between 250 – 350 nm (Figure 4a). As shown in Figure 4, the λ_max for native α₂M was found to be 278 nm, but with increasing concentration of 5-FU, the absorbance increases and a blue shift (10 nm) was observed. The blue shift indicates that the interaction of drug with α₂M causes some structural changes in the native form of protein. It is well-known that 5-FU forms free radicals in the presence of white light that may cause damage or change in the protein. So, quercetin was used as a protective agent to scavenge the free radicals formed by the drug. α₂M was preincubated with quercetin and then treated with 5-FU in the presence of light. When α₂M was preincubated with quercetin, the effect of drug was less compared to drug alone and λ_max was found to be 275 nm (Figure 4b). Our results suggest that quercetin scavenges free radicals formed by drug and prevents oxidative damage and/or restores the native structure of protein.

Figure 5

5-FU was subjected for photo-illumination in order to measure the free radical(s) generated by it. The free radical generated by drug was measured by the NBT reduction assay.Figure 5a depicts the generation of superoxide anions (O₂^.-) with increasing concentration of 5-FU (10 – 100 µM). α₂M was preincubated with quercetin (100 µM) and then exposed to 5-FU for 1 hr in light. According to Figure 5b, quercetin decrease the formation of superoxide anion as compared to 5-FU alone. So, the results suggest that quercetin has antioxidant activity against the formation of free radicals by 5-FU.

Figure 7

Figure 8

The FTIR spectroscopy was utilized to determine the change in the secondary structure of protein. Amide I band was analyzed as it is the most important protein band for the analysis of possible structural changes. The amide I band displays in the wave number range 1600 – 1700 cm^-1 governed by the stretching of the C=O (70 — 85%) and minor contribution of C-N groups (10 — 20%).Figure 6 shows the FTIR spectra of native α₂M along with 5-FU and quercetin. The amide I band for native α₂M was centered at around 1635 cm^-1 which is characteristic of β-sheet structure38. However, upon incubation with 5-FU, the amide I band was at 1635 cm^-1, but the intensity decreases with varying concentration which implies the loss of protein native conformation. The native conformation of α₂M is lost, which affects the functional activity of the antiproteinase inhibitor suggesting that 5-FU causes structural changes in the native conformation of protein, which was confirmed by the antiproteinase activity results. In order to determine the preventive effect of quercetin, the protein was incubated with 5-FU. Figure 6 shows that the band for α₂M preincubated with quercetin was found to be close to that of native protein. Hence, quercetin scavenges the free radicals from the drug and provides a protective role against the oxidative modification of α₂M.

CD spectroscopy was performed to analyse the structural transitions in the structure of protein since it gives information regarding the secondary structural changes49, 50, 51. The CD spectra of α₂M incubated with 5-FU and quercetin are shown in Figure 7. The peak of native protein was found at 215 nm which is a characteristic feature of β-sheet structure38, 39. However, varying the concentration of 5-FU leads to changes in the far UV-CD spectra resulting in the increase, which suggests the loss of β-sheet content in native α₂M. So, the formation of free radicals by the drug causes structural distortion in the native form of protein. Thus, it can be said that the loss in β-sheet content of the protein alters the biological activity of the protein. In order to identify the protective effect of quercetin, it was incubated with α₂M and 5-FU and it was found that α₂M preincubated with quercetin shows spectra very similar to that of native protein.

The effect of drug on the native protein and effect of quercetin on 5-FU treated protein was examined. As depicted inFigure 8a, the native protein shows no fluorescence which indicates no free radicals are present in the sample. 5-FU is known to form free radicals in the presence of white light which can damage the native conformation of protein. When α₂M was incubated with 5-FU (50 µM) it showed bright fluorescence representing the production of free radicals (Figure 8b). When α₂M was preincubated with quercetin and then treated with 5-FU, a weaker fluorescence was observed, which shows a reduction in the formation of free radicals (Figure 8c).