Fluorescence Spectroscopic Analysis of the Interaction of Papain and Bromelain with Procyanidin B3

Xiangrong Li1*, Zhenhua Yang1, Kaiwei Wang2 and Yulin Bai3

1Department of Chemistry, Key Laboratory of Medical Molecular Probes, School of Basic Medicine, Xinxiang Medical University, Xinxiang, Henan, PR China

2School of Physical Education, Xinxiang Medical University, Xinxiang, Henan, PR China

3Grade 2012, Class 25, Clinical Speciality, Xinxiang Medical University, Xinxiang, Henan, PR China

*Corresponding Author:
Xiangrong Li
Department of Chemistry
School of Basic Medicine
Xinxiang Medical University
Xinxiang, Henan, 453003, PR China
Tel: +86-373-3029128
E-mail: 1842457577@qq.com;

Received date: February 24, 2018; Accepted date: March 22, 2017; Published date: April 03, 2018

Citation: Xiangrong Li, Zhenhua Yang, Kaiwei Wang, Yulin Bai (2018) Fluorescence Spectroscopic Analysis of the Interaction of Papain and Bromelain with Procyanidin B3. Genet Mol Biol Res Vol. 1 No. 1:6.

Copyright: © 2018 Li X, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

In this study, the interactions between procyanidin B3 and papain/bromelain were investigated by fluorescence spectroscopy. The results show that the quenching mechanisms are all static quenching at lower concentrations of procyanidin B3, but at higher concentrations of procyanidin B3, maybe predominantly by static-dynamic combined quenching mechanism. The interactions for procyanidin B3 to papain is an enthalpy process and the main intermolecular interactions are van der Waals and hydrogen bond interactions. The binding of procyanidin B3 and bromelain is synergistically driven by enthalpy and entropy, electrostatic and hydrophobic interactions play major roles in the reaction. The binding affinity of procyanidin B3-papain complex is stronger than procyanidin B3-bromelain complex. Synchronous fluorescence spectroscopy shows the interaction between procyanidin B3 and papain/bromelain decreases the hydrophobicity of the microenvironment of tryptophan and tyrosine residues. The study provides an accurate and full basic data for clarifying the binding mechanism of procyanidin B3 with papain/bromelain and is helpful for understanding procyanidin B3’s rational use in the food industry as dietary supplements.

Keywords

Papain; Bromelain; Procyanidin B3; Fluorescence spectroscopy

Introduction

Papain and bromelain, two well-known cysteine-proteases, are widely used in the food, medicine, and pharmaceutical industries and in technical procedures for clinical and laboratory tests [1,2]. Both proteases exhibit structural and chemical similarities such as molecular weight (approximately 23,000), isoelectric point (close to 9.60) and amino acid sequences [3]. The principal differences between the amino acid sequences of papain and stem bromelain are that stem bromelain has only one histidine residue per molecule, whereas papain has two; stem bromelain also contains a methionine residue, while papain does not [4]. Papain can be found naturally in the root, stalk, leaf and fruit of Carica papaya while stem bromelain is derived from the stem of Ananas comosus. Papain plays quite important roles in many biological functions such as proteins cleaving, cells dissociation, necrotic tissue and eczema treatment [5,6]. Moreover, papain has very good efficacy in inflammation, so it has usually been used to treat arthritis and bronchitis. Stem bromelain is widely accepted as a potential phytotherapeutic drug due to its broad medicinal applications such as inhibition of platelet aggregation, angina pectoris, bronchitis, sinusitis, surgical traumas, thrombophlebitis, pyelonephritis and enhanced absorption of drugs particularly antibiotics, analgesic, anti-inflammatory, antitumoral and antituberculosis activity etc. [7-9]. Recent years, the binding of papain with various bioactive molecules have been extensively studied due to their remarkable application prospect in the field of medicine [10-13]. But, as far as we know, the binding of bromelain with bioactive molecules was not reported in the literature.

Proanthocyanidins are naturally occurring compounds that are widely available in fruits, vegetables, nuts, seeds, flowers, and bark. [14]. Besides their participation in food quality attributes such as astringency, bitterness, aroma and color formation [15], proanthocyanidin consumption has been associated with numerous health benefits due to their antioxidant, vasodilatory, anticarcinogenic, antiallergic, antiinflammatory, antibacterial, cardioprotective, immunestimulating, anti-viral and estrogenic activities [16]. Furthermore, proanthocyanidins have been reported to inhibit lipid peroxidation, platelet aggregation, capillary permeability and fragility. They also modulate the activity of enzyme systems including phospholipase A2, cyclooxygenase and lipoxygenase [17]. So they are considered as functional ingredients in botanical and nutritional supplements [18-20].

Proanthocyanidins have the ability to interact with proteins that make them worthy of attention by diverse areas such as medicine, toxicology, chemistry, food science, and agriculture [21].

The binding study between proanthocyanidins and protein had been done previously [21-27]. One of the most widely studied proanthocyanidins is procyanidin B3 (catechin-(4β→8)- catechin; molecular structure: inset of 9 (Figure 1) due to its high abundance in the human diet and relevant antioxidant activity [21]. To our knowledge, an accurate and full basic data for clarifying the binding mechanisms of procyanidin B3 to papain and bromelain were not reported in the literature. In the present study, fluorescence spectroscopy was used as the main source of information on the interaction of procyanidin B3 with papain and bromelain under the physiological conditions.

genetics-molecular-Emission-spectra

Figure 1: Emission spectra of papain and bromelain in the presence of different concentrations of procyanidin B3 at 298 K and pH 7.40. The inset corresponds to the molecular structure of procyanidin B3. The dashed line in the bottom shows the emission spectrum of procyanidin B3 under the same conditions.

Materials and methods

Materials and Instrumentation

Papain and stem bromelain were obtained from Beijing Solarbio Science & Technology Co., Ltd ) and used without further purification. Procyanidin B3 was purchased from Sigma- Aldrich Chemical Company (St. Louis, USA) and used as received. All other reagents were all of analytical reagent grade and were used as purchased without further purification. The pH was determined using a pHS-2C pH-meter (Shanghai DaPu Instruments Co., Ltd, Shanghai, China) at ambient temperature. Sample masses were weighed accurately on a microbalance (Sartorius, BP211D) with a resolution of 0.01 mg. The fluorescence measurements were performed on Cary Eclipse fluorescence spectrophotometer (VARIAN, USA) equipped with a 1.0 cm quartz cell holder and a thermostat bath.

Preparation of Stock Solutions

Double distilled water was used to prepare the phosphate buffered saline (PBS; 0.01 mol L-1 PBS and 0.1 mol L-1 NaCl, pH =7.40). The stock solutions of papain and bromelain were dissolved in a phosphate buffer solution of pH 7.40 and they were stored at 0-4 °C in the dark. Procyanidin B3 was directly dissolved in phosphate buffer solution of pH 7.40. The stock solutions of procyanidin B3 were prepared and used immediately because of oxidation under light and air.

Fluorescence measurements

Certain volume of the stock solution of papain or bromelain (papain and bromelain concentrations were kept at 6.7 × 10-5 mol L-1 and 4.3 × 10-5 mol L-1, respectively) and various volumes of the stock solution of procyanidin B3 were transferred to 0.01 L volumetric flasks in sequence. The mixture was diluted to the experimental concentrations with phosphate buffer solution of pH 7.40 and incubated for 30 min to equilibrate the system. The equilibrated solutions were transferred into the quartz cells for analysis. The corresponding fluorescence emission spectra were recorded over the range of 300-450 nm using an excitation wavelength of 280 nm (where both Trp and Tyr are excited) and 295 nm (where only Trp is excited) at a scan rate of 100 nm min-1. The excitation and emission slit widths were both set at 5 nm. The experiment was measured at four temperatures (293, 298, 303 and 310 K) with recycle water keeping the temperature constant. The synchronous fluorescence spectra were scanned from 290 to 370 nm (Δλ=15 nm) and from 310 to 400 nm (Δλ=60 nm), respectively. The experiments were carried out in triplicates.

In this study, we eliminated the inner filter effect for all of the fluorescence results to obtain accurate data. Therefore, absorbance measurements were performed at excitation and emission wavelengths of the fluorescence measurements. The fluorescence intensity was corrected using the equation [28].

where Fcor and Fobsd are the corrected and observed fluorescence intensities, respectively, whereas Aex and Aem are the sum of the absorbance of protein and ligand at the excitation and emission wavelengths, respectively. The fluorescence intensity utilized in this study is the corrected intensity.

Results and Discussion

Effect of procyanidin B3 on papain/bromelain fluorescence

The effects of procyanidin B3 on the fluorescence intensity of papain and bromelain at 298 K were shown in Figure 1. It can be seen that papain/bromelain has a strong fluorescence emission peak and procyanidin B3 is almost non-fluorescent at λex 280 nm. In all cases, the fluorescence intensity of papain/bromelain decreases regularly with increasing concentration of procyanidin B3. These results suggest that procyanidin B3 can bind to papain/bromelain, and the fluorescence quenching of papain/ bromelain by procyanidin B3 is mainly the concentration dependent. Furthermore, a red shift is observed with increasing procyanidin B3 concentration (Figure 1), which suggests that the fluorophore of papain/bromelain is placed in a more hydrophilic environment after the addition of procyanidin B3 [29]. Same tendencies can be observed at other temperatures (293K, 303K and 310 K).

Upon excitation at 280 nm, both Trp and Tyr are readily excited, while at an excitation wavelength of 295 nm, only Trp emits fluorescence [30]. To determine whether both Trp and Tyr residues are involved in the interaction with procyanidin B3 molecule, the fluorescence of papain/bromelain excited at 295 nm in the presence of procyanidin B3 was also measured. The plots of F/F0 against the concentration of procyanidin B3 are shown in Figure 2, where F0 and F represent the fluorescence intensities before and after the addition of procyanidin B3, respectively. For procyanidin B3, significant difference is observed from the quenching of papain/bromelain fluorescence after excitation at these two wavelengths. These results demonstrate that Trp and Tyr residues are both implicated in the fluorescence quenching.

genetics-molecular-Fluorescence

Figure 2: Fluorescence quenching of papain and bromelain at 298 K and pH 7.40, plotted as extinction of papain and bromelain intrinsic fluorescence (F/F0) against the concentration of procyanidin B3. The fluorescence emission intensity was recorded at λex=280 nm and 295 nm.

Fluorescence quenching mechanisms

There are two types of fluorescence quenching, i.e., dynamic quenching and static quenching. Dynamic quenching increases effective collision number, enhances the energy transfer, and increases the quenching constant of the fluorophore with increasing temperature, whereas static quenching enhances the stability of the compound (ground state complex) formed and thus reduces the quenching constant with increasing the temperature in effect. For fluorescence quenching, the decrease in intensity is usually described by the Stern-Volmer equation [30]

where F0 and F represent the steady-state fluorescence intensities in the absence and presence of procyanidin B3, respectively. KSV is the Stern-Volmer quenching constant and [Q] is the concentration of procyanidin B3. kq is the bimolecular quenching constant, τ0 is the lifetime of the fluorescence in absence of procyanidin B3, assumed to be 10-8 s for biomolecule [31].

Figure 3 shows the Stern-Volmer plots for the papain/ bromelain fluorescence quenching by procyanidin B3. The Stern–Volmer plots are linear at lower quencher concentrations, while the Stern-Volmer plots exhibit an upward curvature, concave toward the y axis at high quencher concentrations. Therefore, to provide a semiempirical measure of the magnitude of the quenching in the procyanidin B3-papain/bromelain systems [32], we discussed the quenching in terms of KSV values computed by linear fits of the Stern-Volmer plots at low quencher concentrations where the plots are nearly linear. The values of KSV obtained from the slope of these plots are listed in Table 1.

genetics-molecular-Stern-Volmer

Figure 3: Stern-Volmer plots of papain and bromelain fluorescence quenched by procyanidin B3 at four different temperatures and pH 7.40.

System T (K) KSV  (L mol-1) kq (L mol-1 s-1)
Procyanidin B3-papain 293 (2.589±0.027)×104 (2.589±0.027)×1012
298 (2.047±0.009)×104 (2.047±0.009)×1012
303 (1.883±0.012)×104 (1.883±0.012)×1012
310 (1.744±0.011)×104 (1.744±0.011)×1012
Procyanidin B3-bromelain 293 (2.535±0.031)×104 (2.535±0.031)×1012
298 (2.509±0.039)×104 (2.509±0.039)×1012
303 (2.475±0.037)×104 (2.475±0.037)×1012
310 (2.333±0.035)×104 (2.333±0.035)×1012

Table 1: The Stern-Volmer quenching constant (KSV) and bimolecular quenching constant (kq) for the interaction of procyanidin B3 with papain and bromelain at four different temperatures and pH 7.40.

Obviously, the KSV values decrease with increasing temperature, and the values of kq are greater than the limiting diffusion rate constant of the biomolecule (2 × 1010 L mol-1 s-1) [33], which illustrates that the quenching mechanism of papain/ bromelain by procyanidin B3 is mainly arisen from static quenching by complex formation in the linear range. The positive deviation in the Stern-Volmer plots at high quencher concentrations may be arising from static-dynamic combined quenching mechanism.

Binding parameters

where F0, F are the same as in Eq. 2. Ka is the apparent binding constant to a set of sites, and n is the average number of binding sites per papain/bromelain. [Q] and [P] are the total quencher concentration and the total protein concentration, respectively. By the plot of log(F0-F)/F vs. log(1/([Q]-(F0-F)[P]/ F0)), the number of binding sites n and binding constant Ka can be obtained (Figure 4 and Table 2).

genetics-molecular-procyanidin

Figure 4: The plots of log (F0-F)/F vs. log (1/([Q]-(F0-F)[P]/F0)) for procyanidin B3-papain system (a) and procyanidin B3- bromelain system (b) at four different temperatures and pH 7.40.

System T (K) Ka (L mol-1) n ΔH (kJ mol-1) ΔG (kJ mol-1) ΔS (J mol-1 K-1)
Procyanidin B3
-papain
293 (1.324±0.051)×105 0.70±0.02 -34.06±0.91 -28.59±0.07 -18.68±2.89
298 (9.460±0.079)×104 0.77±0.04 -28.49±0.05
303 (7.359±0.069)×104 0.86±0.09 -28.40±0.03
310 (6.257±0.077)×104 0.95±0.04 -28.27±0.01
Procyanidin B3
-bromelain
293 (4.721±0.035)×104 0.87±0.03 -13.07±0.13 -26.19±0.01 44.79±0.48
298 (4.236±0.035)×104 0.93±0.07 -26.42±0.01
303 (3.882±0.033)×104 0.99±0.05 -26.64±0.02
310 (3.539±0.034)×104 1.01±0.03 -26.96±0.02

Table 2: The binding parameters and relative thermodynamic parameters for the interaction of procyanidin B3 with papain and bromelain at four different temperatures and pH 7.40.

The decreasing trend of Ka with increasing temperature is in accordance with KSV’s dependence on temperature, which coincides with the static quenching mechanism. The binding constants of papain/bromelain with procyanidin B3 are in the following order as: procyanidin B3-papain> procyanidin B3- bromelain. It is clear that procyanidin B3 interacts with papain with larger binding constant and the binding affinity of procyanidin B3-papain complex is stronger than procyanidin B3- bromelain complex. The values of the stoichiometric binding number n of the two systems approximately equal to 1, suggesting that one molecule of procyanidin B3 combines with one molecule of papain/bromelain and no more procyanidin B3 binding to papain/bromelain occurs at concentration ranges used in this study.

Thermodynamic parameters and binding mode

In general, acting forces between small molecular and biomacromolecule mainly include hydrogen bonds, van der Waals forces, electrostatic interactions, hydrophobic forces, etc. By the values of the binding constants Ka at 293, 298, 303, and 310 K, the thermodynamic parameters such as the free energy change (ΔG), enthalpy change (ΔH) and entropy change (ΔS) were estimated according to the following equations:

where Ka is analogous to the binding constant at the corresponding temperature, and R is the gas constant. The enthalpy change (ΔH) and entropy change (ΔS) are calculated from the slope and intercept of Eq. 4, respectively (Figure 5).

genetics-molecular-Thermodynamic

Figure 5: (a) Van’t Hoff plots of papain and bromelain interaction with procyanidin B3; (b) Thermodynamic parameters of papain and bromelain interaction with procyanidin B3 at 298 K and pH 7.40.

The free energy change (ΔG) is then estimated from Eq. 5. (Figure 5) illustrate the values of the thermodynamic parameters for the interaction of procyanidin B3 with papain and bromelain at 298 K, respectively. Table 2 shows these values at each studied temperature.

From Table 2, it can be seen that the negative values of free energy (ΔG) and enthalpy change (ΔH) support the assertion that the interactions between procyanidin B3 and papain/ bromelain are all spontaneous exothermic processes. The change of Gibbs free energy (ΔG) is the comprehensive embodiment of the changes of enthalpy (ΔH) and entropy (ΔS). The binding of procyanidin B3 to papain is an enthalpy process, while the interaction for procyanidin B3 and bromelain is synergistically driven by enthalpy and entropy. Ross and Subramanian have characterized the sign and magnitude of the thermodynamic parameter associated with various individual kinds of interaction which may take place in protein association process. If ΔH<0, ΔS<0, the main forces are van der Waals and hydrogen bond interactions; if ΔH<0, ΔS>0, electrostatic effect is dominant; if ΔH>0, ΔS>0, hydrophobic interactions play the main roles in the binding reaction [36]. Hence, the main intermolecular interactions may be van der Waals and hydrogen bond interactions in the binding process of procyanidin B3 to papain. For procyanidin B3-bromelain system, electrostatic effect is the major binding force. In addition, the positive entropy (ΔS) value is frequently taken as a typical evidence for hydrophobic interaction. Hence, the main intermolecular interactions may be electrostatic force and hydrophobic interactions in the binding process of procyanidin B3 to bromelain.

Synchronous fluorescence spectra

Synchronous fluorescence spectroscopy technique is successfully applied to explore the microenvironment of amino acid residues by measuring the emission. It offers sensitivity, spectral bandwidth reduction, spectral simplification, and avoiding different perturbing effects [30]. Synchronous fluorescence spectra are obtained by scanning simultaneously the excitation and emission monochromator. The wavelength interval (Δλ) is fixed individually at 15 and 60 nm, at which the spectrum only shows the spectroscopic behavior of Tyr and Trp residues, respectively [37,38]. The effects of procyanidin B3 on the synchronous fluorescence spectra of papain/bromelain are shown in Figure 6. With adding of procyanidin B3, both for papain (Figure 6) and for bromelain (Figure 6), the maximum emission wavelength of Trp is observed to have a red shift along with a red shift of Tyr.

genetics-molecular-Synchronous-fluorescence

Figure 6: Synchronous fluorescence spectra of papain in the presence of different concentrations of procyanidin B3 (Δλ=60 nm (a1) and Δλ=15 nm (a2)) at 298 K and pH 7.40; Synchronous fluorescence spectra of bromelain in the presence of different concentrations of procyanidin B3 (Δλ=60 nm (b1) and Δλ=15 nm (b2)) at 298 K and pH 7.40; Quenching of papain (c1) and bromelain (c2) synchronous fluorescence by procyanidin B3.

This phenomenon expresses the polarity around both Trp and Tyr residues is increased, and thus the hydrophobicity is decreased in the presence of procyanidin B3. In addition, there is another emission at around 380 nm and 365 nm of procyanidin B3- papain/bromelain system at the Δλ=60 nm and 15 nm, respectively. The occurrence of this new band is probably due to the efficient energy transfer from papain/ bromelain to procyanidin B3 [39]. From Figure 6c1 and 6c2, we can see that the curves of Δλ=60 nm are all lower than the curves of Δλ=15 nm, which leads to the conclusion that Trp plays an important role during fluorescence quenching of papain/ bromelain by procyanidin B3.

Conclusion

The binding mechanisms of procyanidin B3 interacting with papain/bromelain were investigated by spectrofluorimetry. Experimental results suggest that procyanidin B3 can bind to papain/bromelain and quench the fluorescence of them. The quenching mechanisms are all static quenching mechanisms at lower concentrations of procyanidin B3, but at higher concentrations of procyanidin B3, maybe predominantly by static-dynamic combined quenching mechanism. The binding constants of papain/bromelain with procyanidin B3 are in the following order as: procyanidin B3-papain>procyanidin B3- bromelain. By evaluating the thermodynamic parameters, it is found that the interactions for procyanidin B3 to papain is an enthalpy process and the main intermolecular interactions are van der Waals and hydrogen bond interactions. The binding of procyanidin B3 and bromelain is synergistically driven by enthalpy and entropy, while electrostatic and hydrophobic interactions play major roles in the reaction. Synchronous fluorescence spectroscopy shows the interaction between procyanidin B3 and papain/bromelain decreases the hydrophobicity of the microenvironment of Trp and Tyr residues.

Acknowledgement

This work was supported by the Doctoral Startup Fund of Xinxiang Medical University (505078), the Foundation for Fostering of Xinxiang Medical University (2014QN122) and the Fund of Fluorescence Probe and Biomedical Detection Research Team of Xinxiang City (CXTD16001).

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