Investigation of the Interaction Between Gallic Acid and α-Amylase by Spectroscopy

Abstract

Gallic acid is one of the important polyphenols in plants and it inhibits α-amylase. The interaction between gallic acid and α-amylase was investigated by fluorescence quenching spectroscopy, UV-vis spectroscopy, synchronous spectroscopy, and the three-dimensional fluorescence spectroscopy under mimic physiological conditions. The result of the emission quenching at different temperatures revealed that there are static quenching of intrinsic fluorescence of α-amylase induced by gallic acid and a complex of gallic acid-α-amylase was formed. The results obtained from the evaluation of three-dimensional fluorescence spectra, UV-vis spectra, and synchronous spectra suggested that the association between gallic acid and α-amylase did change the molecular conformation of α-amylase. Gallic acid can enter the primary substrate-binding pocket and alter the microenvironment around tryptophan and tyrosine residues.

Keywords:

• α-Amylase
• Gallic acid
• Interaction
• Fluorescence quenching
• UV-vis spectroscopy

Introduction

Gallic acid (GA; 3,4,5-trihydroxybenzoic acid), a naturally occurring plant phenol, is abundant in natural plants such as grapes, green tea, different berries, areca nuts, and walnuts.^[1] This compound provides desirable health benefits beyond basic nutrition. Epidemiological evidence suggests that consumption of a diet rich in GA is beneficial to human health, including antioxidant, anti-cancer, anti-inflammatory, and anti-human rhinovirus activities.^[2] It can enhance the cerebral antioxidant defense system and it may be useful in the treatment of vascular dementia.^[1] It is reported that GA produced anti-anxiety, anti-depressant, and antiepileptic effects^[3] in animal models. Moreover, GA can be used to treat human albuminuria and diabetes.^[4] Navita Gupta and others’ study^[5] found that GA was a potent competitive inhibitor of brush border sucrase at acidic pH, it can also inhibited maltase, lactase, and trehalase activities, which may suggest that ingestion of tannins containing GA may interfere in digestive and absorptive functions of mammalian intestinal tracts. As a common dietary constituent, GA may affect the digestive and absorptive functions of the body.

Diabetes mellitus (DM) is a group of metabolic diseases characterized by chronic hyperglycemia resulting from deficiency in insulin secretion or action.^[6] Control of the sugar level in patients’ blood, decreasing the post postprandial hyperglycemia is an effective way to mitigate and prevent the illnesses of hyperglycemic and diabetes from exacerbation.^[7] One practical approach for decreasing postprandial hyperglycemia is to prevent the absorption of carbohydrates after food intake by suppressing of carbohydrate-hydrolyzing enzymes, such as α-amylase and α-glucosidase.^[8,9] α-Amylase (α-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) is an important enzyme in human digestive system and plays an important role in many physiological processes.^[10] It is known that α-amylase is abundant in saliva. Complex polysaccharides are hydrolyzed by α-amylases into dextrins or oligosaccharides at first, and then decomposed into disaccharides and further into monosaccharides by glycosidase when they are taken into human body.^[11] Therefore, α-amylase inhibitors may help to reduce postprandial hyperglycemia by partially inhibiting the enzymatic hydrolysis of complex carbohydrates at the early stage of their digestion, and hence, may delay the absorption of glucose, leading to a lower level of blood sugar for diabetic patients after meals.^[7] Therefore, α-amylase inhibitors can be served as good auxiliary medicines for treatment of diabetes. α-Amylase from porcine pancreas (PPA) consists of 496 amino acid residues, forming a single polypeptide chain with 17 tryptophan (Trp) residues with intrinsic fluorescence. Generally, it is considered that the porcine enzyme is very similar to human α-amylase.^[12]

Natural polyphenols are secondary plant metabolites in response to biotic and abiotic stress. They are the most commonly found chemical compounds in consumed herbal beverages and foods worldwide.^[13] In recent years, natural polyphenols have received much attention because of their potential health benefits^[14] and the study of their mechanism of action has been the subject of considerable interest. It is reported that polyphenols exert protective effect against the development of diabetes, as well as a mitigation effect of diabetes consequences.^[15] GA is one of the most important natural polyphenols. However, to the best of our knowledge, there is limited information on the inhibitory effect of GA on PPA, especially the interaction mechanism, and few efforts have been focused on the molecular binding between them in aqueous solution under physiological conditions in detail. In other words, systematic studies on the interaction between GA and PPA have not been reported up to now.

The inhibitory effect of GA toward PPA has been studied in the present work. Fluorescence spectroscopy has high sensitivity and it can provide information about the molecular environment in the vicinity of the chromophore molecule.^[16] So this methodological approach has been usually used in the study of molecular interactions between ligands and proteins. Hence, this study sought to investigate the details of the interaction between GA and PPA using fluorescence spectrometry (FS), ultraviolet (UV) absorption spectrometry methods. The study has evaluated the quenching mechanism by fluorescence quenching method. The effect of GA on the conformation of PPA involved in the interactions was discussed using UV and the synchronous fluorescence spectrum. Further, a possible modification in the secondary structure of the enzyme has been checked by the three-dimensional spectra fluorescence.

Material and methods

Reagents

α-Amylase (EC 3.2.1.1) from PPA was purchased from Kunming Zero Sci & Tech Co. Ltd., made in German. Dissolubility starch, 3.5-dinitrosalicyclic acid (DNS) and GA were purchased from Kunming Ding Guo Biotech. Co. Ltd. The buffers used in the study were 0.02M sodium phosphate buffer (PBS) at pH 6.9 (with 0.05M NaCl) and 0.05M Tris-HCl buffer at pH 7.4 (with 0.1M NaCl). All the reagents were analytical grade. The water used in all experiments was Milli-Q water.

α-Amylase Inhibition Assay

Briefly, 250 μL GA, with different concentrations, were premixed with 250 μL, 0.02 μM PPA (both were dissolved in 0.02M PBS with 0.05M NaCl), and then incubated at 25°C for 10 min. Then, 500 μL of starch solution (preheated at 25°C in advance) in 0.02 M PBS (pH 6.9 with 0.05 M NaCl) was added to the mixture. Thereafter, the reaction mixture was incubated at 25°C for 10 min. After that, 1.0 mL DNS was added and the reaction was stopped by incubation in a boiling water bath for 5 min and later cooled to room temperature. The reaction mixture was then diluted by adding 8 mL distilled water and the absorbance was measured at 540 nm in a spectrophotometer. The reference sample included all other reagents and the enzyme with the exception of the test sample. Appropriate solvent controls were maintained.^[17,18] The PPA inhibitory activity was expressed as percentage inhibition, and it can be calculated as following equation:

(1)

where A_ref is the absorbance of the reference; A_sample is absorbance of the test samples.

Fluorescence Measurements

Fluorescence quenching

The fluorescence intensities were recorded with a Perkin-Elmer LS 50 Luminescence Phosphorescence Spectrophotometer. The quenching effect of GA with different concentrations on PPA was assayed as described in the literature with some modifications.^[19,20] A certain amount of amylase (with a concentration and volume of 2×10⁻⁶ M and 1.0 mL, respectively) was dissolved in Tris-HCl buffer (0.05 M, pH 7.4, with 0.1 M NaCl to keep the same ion intensity). The GA solutions were dissolved in the same Tris-HCl buffer. After addition of different concentrations of GA (40 to 200 μM) to α-amylase solution, the GA and α-amylase were mixed and incubated for 2 h at different temperatures (301 and 313 K) and then their emission spectrum were recorded. Both slit widths of the excitation and emission were 10 nm. In order to get the appropriate excitation wavelength, we recorded the emission spectrum of the pure enzyme from 290 to 450 nm at different excitation wavelength (270 to 295 nm). The results showed that for the same concentration, the enzyme has the greatest intensity when the excitation wavelength was 278 nm. Therefore, the excitation wavelength of 278 nm was used to follow the PPA fluorescence. All the data were recorded at the range of 290 to 450 nm.

Synchronous fluorescence

The synchronous fluorescence spectra of PPA in absence and presence of different concentrations of GA were recorded. The D-values between the excitation and emission wavelength (Δλ) was set at 15 and 60 nm^[21] and the emission wavelength was set from 240 to 340 nm and 240 to 320 nm, respectively. Other parameters were the same as that of the fluorescence quenching spectra.

Three-dimensional fluorescence spectroscopy

In addition to fluorescence quenching and Synchronous fluorescence, the study has recorded the three-dimensional fluorescence spectroscopy of enzyme and the GA-PPA compound system. The three-dimensional spectroscopy is the total luminescence spectroscopy, which is the simultaneous measurement of excitation, emission, and intensity wavelengths of compound fluorophores. The three-dimensional spectroscopy often serves as a unique method to study a single compound or a mixture of fluorescence components.^[22] This spectrum can inform about conformational changes of PPA by the interaction with GA. The emission wavelength range was selected from 200 to 410 nm, the initial excitation wavelength was set to 200 nm with increment of 10 nm, and the scanning number was 15. Other parameters were the same as that of the fluorescence quenching spectra.

UV Absorption Spectrum

The UV absorbance spectra of PPA, GA, and PPA-GA system were carried out at 301 and 313 K (pH 7.4) equipped on a ULTROSPEC 2100 PRO with 1.0 cm quartz cells. Keep the concentration of PPA solution at 2×10^–6 M and add different volume of GA to make the final concentration of GA as 0.0, 40, 60, 80, 100, 150, 200 μM. The UV absorption spectrum was recorded in the wavelength range of 190 to 310 nm. The experiments were carried at 301 and 313 K. Appropriate solvent controls were maintained.^[21]

Results and Discussion

Effect of GA on α-Amylase

In order to explore the effect of GA on the activity of PPA, we calculated the inhibitory ratios of different concentration of GA on PPA. Fit the points into a curve. The curve-fitting equation is:

As shown in Fig. 1, the PPA inhibition ratio increased with the incremental addition of GA, indicating that the activity of the enzyme decreased by GA gradually. When the concentration of GA reached 10 mg/mL, the inhibition ratio of GA on PPA increased to 49.22%. Ye and others^[23] found that the inhibition ratio of rutin, vanillic acid, ferulic acid, betulinic acid, and chlorogenic acid on α-amylase are 9.6, 2.2, 7.0, 53.2, and 35.5%, respectively. We can say that GA can serve as a good inhibitor for α-amylase.

FIGURE 1 Influence of gallic acid on the activity of α-amylase ([gallic acid] is the concentration of gallic acid).

Fluorescence Quenching

Information of the binding between small molecules and protein can be provided by fluorescence measurements. The intrinsic fluorescence of proteins can provide considerable information about their structure and dynamics, and it is often considered on the study of protein folding and association reactions.^[24] In a protein molecule, Trp is the principal residue responsible for the fluorescence intensity. Changes in emission spectra of Trp are commonly in response to protein conformational transitions, subunit association, substrate binding, or denaturizing.^[25] Since PPA contains 17 Trp residues with the major fluorescence intensity in the molecule^[12] and they are quite sensitive to the surrounding medium, we can track the change of PPA’s conformation in response to polyphenol binding by taking Trp as the intrinsic fluorescence probe.

In the present work, the interaction between GA and PPA were followed by the changes in the fluorescence emission spectra of PPA in the absence and presence of the polyphenols. The quenching fluorescence measurements were carried out to obtain detailed information about the interaction between the ligand and the protein. Figure 2 shows the fluorescence emission spectra (λ_ex = 278 nm) of PPA at pH 7.4, 301 K (A) and 313 K (B) with the addition of GA as quencher, whose concentration ranges from 0 to 200 μM. We can get the information about the wavelength at maximum emission intensity (λ_max) and the fluorescence intensity at the λ_max from the curves. As can be seen in Fig. 2, at pH 7.4, there is a fluorescence peak at near 345 nm. Increasing concentrations of GA leads to a regular quenching of α-amylase fluorescence. Thus, GA can bind to α-amylase and quench the Trp intrinsic fluorescence intensity. Under the temperature of 301 K (Fig. 2a), an obvious shift in λ_max form 344 to 353.08 nm toward higher wavelengths (called red shift) is observed with the addition of GA and it is from 346.5 to 353.38 nm for 313 K (Fig. 2b). It is reported that the fluorescence spectrum of proteins that contain Trp residues mainly display the fluorescence characteristics of Trp, whose λ_max is between 310 to 360 nm. The λ_max for Trp residues located on the protein surface is 350–353 nm; however, the λ_max for the ones located in the nonpolar region, i.e., the ones buried in a hydrophobic cavity is 326–342 nm.^[26] From the changes in the fluorescence spectrum in Fig. 2, we can speculate that the peak belongs to tyrosine (Tyr) residues of α-amylase, which is buried within protein interior and in a relatively nonpolar environment. After interacted with GA, the surroundings of Trp residues are exposed to water and become more polar. That is to say the hydrophobic cavity in PPA is disagglomerated and the structure is loosened.^[27] The fluorescence quenching data indicate that there are binding interactions between PPA and GA.

FIGURE 2 Emission spectra of 2 μM α-amylase in the presence of gallic acid (excitation wavelength (λ_ex) = 278 nm; pH 7.4 at (a) 301 K and (b) 313 K; curves 1 to 7: concentrations of gallic acid ([gallic acid]) = 0, 40, 60, 80, 100, 150, and 200 μM, respectively).

Fluorescence quenching is the decrease of the quantum yield of fluorescence from a fluorophore induced by a variety of molecular interactions with a quencher molecule.^[28] Fluorescence quenching can be dynamic, resulting from collisional encounters between the fluorescent molecules and the quencher, which does not affect the structure and the biological activity of the protein, or static, resulting from the formation of a ground state between the protein and the quencher so that leads to the change of secondary structure, and therefore, affect the physiological activities of the protein.^[26] The mechanism can be distinguished by the quenching rate constant which is dependent on temperature. For static mechanism, the quenching rate constant decreased with the increased temperature and the opposite effect for the case of dynamic quenching.^[29] In both cases, molecular contact is required between the fluorophore and the quencher for fluorescence quenching to occur.^[30] Fluorescence quenching can be described by the Stern-Volmer equation (Eq. 2).

(2)

In the above equation, F₀ and F are the fluorescence intensities in the absence and presence of the quencher, respectively. Kq is the bimolecular quenching constant of protein, Ksv is the Stern-Volmer quenching constant, τ₀ is the lifetime of the biomolecule in the absence of the quencher (τ₀ = 10⁻⁸s)^[31] and [Q] is the concentration of the quencher. Hence, Eq. (2) was applied to determine Ksv and Kq by linear regression of a plot of F₀/F against [Q]. A linear Stern-Volmer plot (curve of F₀/F versus [Q]) is generally indicative of a single class of fluorophores in a protein, all equally accessible to the quencher. It also means that only one mechanism of quenching occurs (dynamic or static).^[32] However, if the quenching type is combined quenching (both static and dynamic), the Stern-Volmer plot has an upward curvature, concave toward the y-axis.^[33]

To interpret the data from fluorescence quenching studies, it is important to understand what kind of interaction occurs between the quencher and the fluorophore. The Stern-Volmer plots of the fluorescence quenching of PPA by GA are shown in Fig. 3. The plots are linear and the linearity is independent on the temperature, which indicates that there may be a single type of quenching, either static or dynamic in the binding interaction of α-amylase with GA.^[34] In the cases of a static mechanism, there is a complex formation, and in such cases, the bimolecular quenching constant (Kq) is calculated.^[16] Kq can reflect the efficiency of quenching or the accessibility of the fluorophores to the quencher and it allows one to verify if the quenching is due to a complex formation with proteins that affect Trp’s microenvironment. From the experimental data, we get the following equations by liner regression fitting:

FIGURE 3 The Stern-Volmer plot for α-amylase fluorescence quenching caused by gallic acid at 301 and 313 K. (In all cases, excitation wavelength (λ_ex) = 278 nm, F₀, and F are the fluorescence intensities in the absence and presence of gallic acid, respectively, [gallic acid] is the concentration of gallic acid.)

The Kq obtained at each temperature are calculated according to Eq. (2) and indicated in Table 1. It is known that the maximum scatter collision quenching constant in water for a dynamic mechanism is 2.0×10¹⁰ L·mol^–1·S^–1.^[35] As can be seen in Table 1, the bimolecular quenching constant (Kq) obtained for α-amylase quenching caused by GA are greater than rate constant limit for dynamic quenching, indicating that the quenching mechanism may be static type with great probability. Besides, the quenching constants decreased with an increase in temperature, a characteristic for static quenching, due to that high temperatures result in the dissociation of complexes formed.^[30] The results suggest that the quenching mechanism of GA with PPA is static style and GA can combine with PPA to form PPA-GA complexes.

TABLE 1 Bimolecular quenching constants (Kq) for the interaction of gallic acid with α-amylase at different temperatures

pH	T (K)	Kq (10^12L·mol^–1·S^–1)	R^2
7.4	301	1.155	0.9945
	313	0.915	0.9874

The binding parameters of PPA with GA are very important to understand the interaction of GA with PPA. In order to characterize the binding model of α-amylase with GA, such as binding constants (Ka) and binding sites (n) are also analyzed by Eq. (3) as following:

(3)

In the above equation, [Q] is the GA concentration, Ka is the binding constant, and n is the number of binding sites, T is the absolute temperature. The plot of log[(F₀ – F)/F] versus log[Q] is showed in Fig. 4, which gives the number of binding sites and the binding constant from the slope and intercept. The binding sites and the binding constant are listed in Table 2. In the range of temperature studied, the value obtained for n, near to 2, indicates that the bind of GA to α-amylase is in a ratio near 2:1. From the Ka value it can be deduced the formation of a relatively stable complex.^[29] The Ka value decreased with the increase of the temperature due to that high temperature go against the formation of the complex, which shows that the quenching mechanism between GA and PPA is static quenching.

TABLE 2 The binding constant (Ka) and binding sites (n) of gallic acid with α-amylase at different temperatures

T (K)	Ka (10^3L·mol^–1)	n	R^2
301	0.836	1.8543	0.9868
313	0.963	1.7042	0.9984

FIGURE 4 Plot of log [(F₀ – F)/F] = log Ka + n log [Q] for determination of binding parameters at different temperatures. (Other conditions are the same as in Fig. 3, [Q] is the concentration of gallic acid, F₀, and F are the fluorescence intensities in the absence and presence of gallic acid, respectively. Ka is the binding constant, n is the binding site, [gallic acid] is the concentration of gallic acid).

Synchronous Fluorescence

In order to explore the microenvironment changes of PPA by addition of GA, we measured synchronous fluorescence spectra of PPA with GA. In synchronous fluorescence spectroscopy, both the excitation and emission monochromators are scanned simultaneously in such a manner that a constant wavelength interval is kept between the emission and excitation wavelength (Δλ).^[36] Synchronous fluorescence has many advantages in studying the effect of ligand on large molecules such as enzyme. It has good selectivity and high sensitivity. It can also narrow the bandwidth and avoiding different perturbing effects.^[28] Selecting the appropriate wavelength difference can separated the overlapping fluorescence peak on the ordinary fluorescence spectrum. Yuan and others^[37] suggested that the shift in position of λ_max in synchronous fluorescence spectra is corresponding to the changes of the polarity around the chromosphere molecule which can study the environment of amino acid residues in protein. Studies have showed that when the D-values between the excitation and emission wavelength (Δλ) are stabilized at 15 or 60 nm, the synchronous fluorescence spectra give the spectral characteristic information of Tyr or Trp residues, respectively.^[21] In order to study the alteration in the microenvironment around the Tyr and Trp residues in the secondary structure changes induced by GA, the interaction of GA with the residues of PPA were analyzed by synchronous fluorescence spectra. The effect of GA on PPA synchronous fluorescence is shown in Fig. 5.

FIGURE 5 Synchronous fluorescence spectrum of 2 μm α-amylase; pH = 7.4; curves 1 to 7:[gallic acid] = 0, 40, 60, 80, 100, 150, and 200 μM, respectively. (a) Δλ = 15 nm; (b) Δλ = 60 nm ([gallic acid] is the concentration of gallic acid, Δλ is the wavelength interval between the emission and excitation wavelength).

From Fig. 5, when Δλ = 15 nm, the wave shape changes obviously with the addition of GA, indicating that there are conformational changes near the Tyr residues. However, when Δλ = 60 nm, the shape of the wave changed inconspicuously, indicating that the conformational near Trp didn’t change greatly. Under both of the Δλ, there are conspicuous red shift of the λ_max when GA was gradually added into the PPA, suggesting that the polarity of the microenvironment around the aromatic residues buried in nonpolar hydrophobic cavities are changed by GA, both of Trp and Tyr residues were moved to a more hydrophilic environment and more exposed to the solvent.^[38] It may be concluded that GA can bind PPA and induces the tertiary structure changes of PPA. The microenvironment around the aromatic amino acid residues was changed. Besides, the fluorescence intensity of Trp (Fig. 5b) is much more than Tyr (Fig. 5a), which indicate that Trp contribute the main fluorescence intensity to PPA.

Three-Dimensional Fluorescence Spectroscopy

As already known, the binding of a protein and ligand is often coupled to a structural change in the protein that makes the binding site more complementary to the ligand, permitting tighter binding, so conformational alterations of proteins are a factor of biological function. The three-dimensional fluorescence spectrum is a developed technology for fluorescence analysis in the recent 20 years. It’s the total luminescence spectroscopy that measures the excitation, emission wavelengths, and intensity of compound fluorophores simultaneously.^[21] The three-dimensional fluorescence spectrum has high measurement sensitivity and good selectivity for the molecular structure and it can fully demonstrate the synthetically fluorescent information of the samples.^[33] Hence, this method often serves as a unique method to study a single compound or a mixture of fluorescence components.^[22] The three-dimensional fluorescence spectra and contour of PPA and GA-PPA compounds are shown in Fig. 6 and the corresponding parameters are collected in Table 3.

TABLE 3 Three-dimensional fluorescence spectral characteristic parameters of α-amylase and α-amylase-gallic acid system

System	Peak 2 λex^a/λem^b (nm/nm)	Δλ^c (nm)	Intensity	Peak 3 λex/λem (nm/nm)	Δλ (nm)	Intensity
α-amylase	280/348	68	94.113	220/348	128	180.33
α-amylase+gallic acid (1:2)	280/350.05	70.05	49.705	220/348	128	107.09

^aλex is emission wavelength;

^bλem is excitation wavelength;

^cΔλ is the wavelength interval between the emission and excitation wavelength.

FIGURE 6 Three-dimensional fluorescence spectra of α-amylase (a) and α-amylase-gallic acid system (b). [α-amylase] = A: 2 μM; B: 2 μM; [gallic acid] = A: 0 μM; B: 4 μM ([α-amylase] is the concentration of α-amylase, [gallic acid] is the concentration of gallic acid).

Figure 6 presents the three-dimensional fluorescence spectrum (A-1, B-1) and the contour spectra (A-2, B-2, of PPA (a) and PPA-GA system (b). From the figure, we can see mainly two kinds of fluorescence peaks, the Rayleigh scattering peak (Peak 1) and the intrinsic fluorescence of Trp and Tyr residues (Peak 2 and 3). The increasing “ridge shape of the peak” (peak 1) in A-1 and B-1 is corresponding to the “pencil lines” in A-2 and B-2. They are corresponding to Rayleigh scattering, which characterized by λ_em = λ_ex.^[22] The “camel shape” of the peak (peak 2 and peak 3) in A-1 and B-1 is corresponding to the “fingerprint lines” in A-2 and B-2, respectively. With the increasing of the excitation wavelength from 200 to 350 nm (the increment is 10 nm), the λ_em of peak 2 and peak 3 remained the same (near 345 nm), this is the typical characteristic of the fluorescence peak. The two peaks are the typical intrinsic fluorescence of Trp and Tyr residues in PPA.^[39] Peak 2, centers at 280/348 nm (λ_ex/λ_em), mainly represents the spectral feature of Trp and Tyr residues of PPA involving π-π transition and reflects changes in the tertiary structure of the protein. Peak 3, centers at 220/348 nm (λex/λem) mainly exhibits the spectral characteristics of the microenvironment of polypeptide backbone structure C=O which is caused by the n-π* transition, it is related to modifications of secondary structure of PPA.^[22] Analyzing from the intensity changes of the peaks in the figure, all of them decrease obviously in different degree after the addition of GA (Table 3). The fluorescence intensity of peak 2 decreases from 94.113 to 49.705 (decreased about 47.2%) and peak 3 decreases from 180.33 to 107.09 (decreased about 40.05%) in presence of GA, meaning that the adduct of PPA and GA induced the great destabilization of the polypeptide chain of PPA, which increased the exposure of some hydrophobic regions that had been buried before.^[22] The fluorescence intensity ratios of peak 2 and peak 3 are 1.91:1 and 2.15:1 in absence and presence of GA, respectively. Due to the need of avoiding the water, the hydrophobic group and side chains are forced to fold. This kind of folding tends to embed the hydrophobic residues within the molecule to form hydrophobic cavity. It is speculated that the combining site of GA and PPA is in such a hydrophobic cavity because the fluorescence residues such as Trp and Tyr contain large hydrophobic side chains.^[40] The binding site is near the Trp and Tyr residues through the π-π stacking between aromatic rings of GA and phenyl rings of Trp or Tyr residues.^[21] The decrease of fluorescence intensity of PPA for both peaks indicate that the interaction with GA induce a conformational modification of PPA and changes the hydrophobic microenvironment polarity of the binding site. The structural changes detected via three-dimensional fluorescence are in good conformity with fluorescence quenching data previously, a complex is formed between GA and PPA.

UV Spectrum

UV-vis absorption measurement is a very simple method. It is applicable to explore the structural changes and to know the complex formation. The UV absorption spectrum is mainly caused by electronic excitation of aromatic amino acids like Trp and Tyr, followed by phenylalanine (Phe) and Histidine (His),^[41] and they are quite sensitive to the change of molecule’s conformation which can reflect the tiny structure vibration of protein molecular.^[42] The UV absorption spectra of PPA solution (2 μM) and PPA-GA system are displayed in Fig. 7. As can be seen in Fig. 6, there are two absorption peaks at 211 and 260 nm, respectively on the PPA absorption spectrum, which is corresponding to peptide bond and the aromatic amino acid absorption, respectively.^[33] The addition of GA produces an increase in the intensity of UV absorption, accompanied by an obvious red shift of the maximum absorption peak near 211 nm (from 211 to 221 nm). As can be seen in the figure, the absorption spectra changes induced by the interaction of GA with PPA, indicating that there are static quenching with complex formation at ground state between GA and PPA as the dynamic quenching only affects the excited states of the fluorophores and the absorption spectra does not change.^[21] This agrees with the result of previous fluorescence quenching analysis.

FIGURE 7 Absorption spectra of 2 μm α-amylase in the presence of gallic acid; pH = 7.4; curves 1 to 7: [gallic acid] = 0, 40, 60, 80, 100, 150, and 200 μM, respectively ([gallic acid] is the concentration of gallic acid).

The absorption band of protein near 210 nm is caused by the electron displacement transition of peptide bonds, it represents the spiral content of α-helix structure of the protein.^[43] The absorption of PPA near 250 nm is mainly due to the π-π* conjugate system electron transitions of peptide bond C=O in Trp and Tyr residues and it is related to the quantity of α-helix.^[44] The increase of the PPA spectra with the addition of GA indicates that GA is favor for the union between and within the PPA molecules and then the conformation of PPA changed. This change, on the one hand, leads to the decrease of α-helix quantity and the intramolecular effect.^[45] On the other hand, the π-π* electronic transition energy gap (ΔE) decreases and the probability of electronic transition increases. Both of the two effects can lead to the increase and the red shift of the absorption peaks.^[46] These phenomena indicate that there are binding interactions between GA and PPA. GA enters into the hydrophobic pocket of PPA to form complexes with PPA and the microenvironment of Trp and Tyr residues become more polar. That is to say the peptide strands of PPA are loosened partly and unfolded and the aromatic amino acids are exposed to a more hydrophilic region. The conclusion agrees with the result of conformational changes by synchronous fluorescence spectra, which indicates that the approach of synchronous fluorescence spectroscopy is scientific.

Conclusions

To conclude, GA can inhibit the activity of PPA. The interaction between GA and PPA has been investigated by spectroscopic methods including fluorescence spectroscopy and UV-vis absorption spectroscopy. The fluorescence quenching study reveals that the quenching mechanism of GA with PPA is static quenching and a complex is formed between them. The binding interaction should be located near or at the catalytic site. The synchronous fluorescence, three-dimensional fluorescence and UV-vis spectra data also indicate that binding of GA to PPA can induce conformational changes in the secondary structure of the enzyme, the microenvironment around Trp and Tyr residues has been changed. The present study will increase some useful knowledge in the fields of green medicine on decreasing the levels of sugar in human blood.

FUNDING

We gratefully acknowledge financial support of the National Natural Science Foundation of China (Grant No. 21466037), Supporting Plan Issue of the Ministry of Science and Technology of China (2011BAD46B00), and the Science and Technology Major Program of Yunnan Education Bureau (ZD2014009).

Additional information

Funding

We gratefully acknowledge financial support of the National Natural Science Foundation of China (Grant No. 21466037), Supporting Plan Issue of the Ministry of Science and Technology of China (2011BAD46B00), and the Science and Technology Major Program of Yunnan Education Bureau (ZD2014009).