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Influence of UV radiation on molecular structure and catalytic activity of free and immobilized bromelain, ficin and papain
Journal of Photochemistry & Photobiology, B: Biology 201 (2019) 111681
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Influence of UV radiation on molecular structure and catalytic activity of free and immobilized bromelain, ficin and papain
T
Marina Holyavkaa, , Svetlana Pankovaa, Victoria Korolevaa, Yuliya Vyshkvorkinab, Anatoliy Lukina, Maxim Kondratyevc, Valeriy Artyukhova ⁎
a
Voronezh State University, Universitetskaya sq. 1, Voronezh 394018, Russian Federation Moscow Institute of Physics and Technology (National Research University), Institutskiy per. 9, Dolgoprudny, Moscow Region 141700, Russian Federation c Institute of Cell Biophysics of the Russian Academy of Sciences, Institutskaya st. 3, Puschino, Moscow region 142290, Russian Federation b
ARTICLE INFO
ABSTRACT
Keywords: UV irradiation Adsorption immobilization Bromelain Ficin Papain Chitosan
Our research has shown that the degree of photosensitivity of the cysteine proteases can be arranged in the following order: bromelain → ficin → papain. After the UV irradiation with 151 J·m−2 intensity of a bromelain solution, the enzyme activity has increased. No decrease in the catalytic capacity and the change in the size of the molecule was recorded in the 151–6040 J·m−2 range of irradiation intensities. A decrease in the catalytic capacity of ficin and the increase of its globule size occurred after exposure to a radiation of 3020 J·m−2 intensity. The decrease in papain activity was observed at the UV irradiation intensity of 453 J·m−2, and an increase of the papain globule size was detected at 755 J·m−2. Immobilization on chitosan matrix leads to the increase in the stability of heterogeneous biocatalysts with respect to UV irradiation in comparison with free enzymes. The changes in IR spectra of immobilized cysteine proteases practically do not affect the bands due to the protein component of the system: amide I, amide II, amide III. Therefore, it can be postulated that the chitosan matrix acts as photoprotector for immobilized ficin, bromelain and papain. The obtained results can be helpful for development of drugs based on chitosan and cysteine proteases in combination with phototherapy, as well as for choosing their sterilization conditions.
1. Introduction UV irradiation is one of the generally recognized factors that have a complex impact on organisms at both cellular and molecular levels. Effects from ultraviolet irradiation are initiated by the photochemical absorption of light quanta by molecules of biological significance. The most important of them are nucleic acids, proteins and, in much less degree, some other molecules [1]. UV irradiation can initiate the development of photodermatoses and provoke the development of preand neoplastic processes in human skin. In addition, under the influence of prolonged exposure to UV irradiation, a whole complex of involutive biological processes in various skin layers take place, e.g. photoaging, the clinical manifestation of which is dermatogeliasis [2,3]. Of a particular interest is the study of molecular mechanisms of phototherapy, which is a highly effective option for the treatment of skin diseases, for example, beneficial effects of UV irradiation have
been proven for atopic dermatitis. This method allows to normalize the content of key pro- and anti-inflammatory cytokines in the patients, as UV rays have an impact on the production of immune cells (cytokines), possessing immunosuppressive and anti-inflammatory effects [4]. UV irradiation can influence on activity of various phagocytic cells (neutrophils and dendritic cells), oxidation of blood cell lipids, and various functions of erythrocytes and leukocytes [5]. Its therapeutic effect is based on photochemical reactions that influence on surface receptors and protein (enzymatic) systems of immunocompetent cells. UV irradiation is used to stimulate the components of the immune system and, possible, to exploit the activated immunocytes [3,4]. In addition, the influence of UV irradiation on biomaterials should be investigated systematically, since it is often used for drug sterilization in medical practice [6,7]. Various proteases may act as antimicrobial agents in the treatment of skin lesions. Proteolytic enzymes play a role in several physiological processes: from protein maturation and its degradation to more specific
Corresponding author. E-mail addresses: [email protected] (M. Holyavka), [email protected] (Y. Vyshkvorkina), [email protected] (A. Lukin), [email protected] (V. Artyukhov). ⁎
https://doi.org/10.1016/j.jphotobiol.2019.111681 Received 17 February 2019; Received in revised form 13 October 2019; Accepted 30 October 2019 Available online 01 November 2019 1011-1344/ © 2019 Elsevier B.V. All rights reserved.
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regulation functions. Proteases are the most widely and effectively used enzymes in the food and pharmaceutical industries. They amount about 60% of the global sales of enzymes. Only a few of plant proteases are used industrially. Papain, bromelain and ficin are the most commercially important endopeptidases from plant sources [8,9]. Bromelain (EC 3.4.22.4) is a high molecular weight glycoprotein; its highest content is found in the green pineapple juice. Bromelain is the proteolytic enzyme similar to pepsin and papain, since it breaks down proteins to poly- and oligopeptides. The hydrolysis of these proteins occurs at a wide pH range from 3.0 to 8.0. The molecular weight of bromelain is about 33 kDa; its molecule forms a single polypeptide chain containing 285 amino acid residues. Bromelain has an immunomodulating effect, accelerates tissue repair processes resulted from depolymerization of intercellular structures and modification of vascular permeability. The anti-inflammatory and anti-aggregative effects of bromelain are due to its ability to change the metabolism of arachidonic acid [8,10]. Ficin (EC 3.4.22.3) is a proteolytic enzyme from the latex of the plants genus Ficus. Enzyme refers to cysteine proteases, has a rather wide substrate specificity and shows high activity in the pH range from 6.5 to 9.5. The molecular weight of ficin is 25–26 kDa. The molecule consists of one polypeptide chain. The enzyme preferably hydrolyses the tyrosine and phenylalanine bonds, while neither arginine nor lysine bond splitting has been observed at all. Ficin has antimicrobial activity against Gram-positive and Gram-negative bacteria [8]. Papain (EC 3.4.22.2) is a monothiol cysteine endoprotease. It is active not only in acidic, but also in neutral and alkaline media: pH range from 3.0 to 12.0 (optimum pH 6–7). The molecular weight of papain is 23 kDa. It is a protein consisting of 212 amino acid residues. Papain accelerates the healing of wounds, trophic ulcers, and decubitus by promoting their purification from necrotic masses, accelerates the cleansing of the wound from pathogenic microflora, creates optimal conditions for reparative processes. In biological and biomedical scientific research, papain is widely used in studying the receptor apparatus of cells, the structure of proteins, chemical analysis and purification of various biologically active substances [8,10]. The main disadvantage of the soluble form of proteolytic enzymes is their rapid inactivation due to proteolysis [11,12]. One of the ways to increase the stability of enzymes is their immobilization on polymeric carriers [13,14]. Promising carriers for the immobilization of protein drugs are chitosans and their derivatives, which have antibacterial activity, low allergenicity and relatively low cost [15–21]. In addition, there have been attempts to test chitosan as a carrier for photosensitizers such as porphyrazine. Chitosan effectively prevents photodegradation of their macrocycles [22]. It also forms porous structures and thin films used in cell transplantation and tissue regeneration. In medicine and pharmacy, chitosan is used as a part of bandages, sponges, membranes, artificial leather, contact lenses, funds for the treatment of bone diseases and surgical sutures [23–25]. The properties of chitosan can be modified by adding another polymer [26–30], as well as by the influence of ultraviolet irradiation, mechanical treatment and heating [31–33]. At present, the problem of creating highly effective healing agents with prolonged action for burns and purulent necrotic wounds is not solved. A reason is a long list of requirements imposed to such drugs: non-allergenicity, nontoxicity, biocompatibility and biodegradability, the need to remove the wound exudate, prevention of the wound surface contamination, protection from mechanical damage and drying, possibility for acceleration of skin healing by introducing additional pharmaceuticals substances, ability to maintain the therapeutic effect throughout the storage period. In connection with the emergence and increased reproduction of antibiotic-resistant bacteria, UV irradiation in conjunction with cysteine proteinase therapy should be investigated as an alternative approach to the infection treatment. The complex usage of the UV irradiation, cysteine proteases and chitosan may help to restore the wound or burn faster and to shorten the skin healing time.
Our aim was to study the effects of UV irradiation on photomodulation of bromelain, ficin, papain activity, both free and immobilized on chitosan matrix. 2. Materials and Methods UV irradiation of the free and immobilized cysteine proteases was carried out by their continuous mixing with a magnetic stirrer in a thermostated cell (20 ± 1 °C) using a mercury-quartz lamp of the DRT400 type through a UVS-1 filter with a transmission band of 240–390 nm for 1, 3, 5, 10, 20, 30, 40 min. The intensities of irradiation were 151, 453, 755, 1510, 3020, 4530 and 6040 J·m−2 respectively. The spectral distribution of the DRT-400 lamp's radiation passed through the UVS-1 filter is shown in Fig. 1S of supplementary material. The cysteine proteases – bromelain (B4882), ficin (F4165) and papain (P4762) – were purchased from Sigma-Aldrich, as well as bovine serum albumin (BSA) which served as a substrate for hydrolysis. Two types of acid-soluble chitosan, the medium-molecular weight (200 kDa) and high-molecular weight (350 kDa), were synthesized by Bioprogress Ltd. The UV spectra of the studied systems are shown in Fig. 1S of supplementary material. Immobilization of proteases on the chitosan matrix was carried out by the adsorption method [34]. The catalytic activity of enzymes was determined by the modified Lowry method. Catalytic activity and protein content are presented in Table 1S in Supplementary material. The enzyme amount hydrolizing 1 μM of bovine serum albumin (BSA) per 1 min was taken as a unit of protease activity. The enzymes' molecular size was determined by dynamic light scattering method using the Nano Zetasizer ZS equipment (Malvern Instruments). The back-scattered light from 4 mW He/Ne laser at 632.8 nm wavelength was focused at an angle of 173°. The protein concentration was 1.0–1.5 mg mL−1 in a 0.1 M phosphate buffer at рН 7.4 and a temperature of 25°С. The preparation was passed through a Millipore filter with pores of 0.45 μm in diameter. Registration of infrared spectra of the analyzed samples was carried out on the Bruker Vertex-70 IR-Fourier spectrometer. The spectra were recorded from non-oriented powder samples. Unfortunately, our instrumentation does not allow to measure reduction of the fluorescence lifetime or quantum yield of enzymes after immobilization on chitosan matrix. The amino acid sequences of the studied proteases were aligned using the BioEdit software package. Visualization of chromophores was carried out in the Maestro program. The enzyme structures were prepared for docking according to the standard scheme for Autodock Vina package: the atoms (together with their coordinates) of the solvent, buffer and ligand molecules were removed from the PDB input file. Before carrying out the numerical calculations, a charge was placed on the surface of proteins using MGLTools. The center of the molecule and the parameters of the box («cells») were set manually, ensuring that the whole protease molecule was placed within the computational area of space. The chitosan structure model was drawn in the HyperChem molecular designer; this structure was consistently optimized first in the AMBER force field, and then quantum-chemically in PM3. The ligands in the docking calculations had the maximum conformational freedom: rotation of the functional groups around all single bonds were allowed. Charge arrangement on the chitosan molecule and its protonation/deprotonation was performed automatically in the MGLTools 1.5.6 package. 3. Results and Discussion 3.1. Photosensitivity of Free and Immobilized Bromelain With UV irradiation of bromelain solution at intensity of 151 J·m−2, the enzyme activity was increased by 86% compared to the control 2
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Fig. 1. Effect of UV irradiation on the specific catalytic activity (left), diameter of the enzyme molecules (middle) and their variation in % (right). In the left graph, black color is used for the free enzyme, gray for the enzyme immobilized on medium molecular weight chitosan, white for that immobilized on high molecular weight chitosan. In right graph, gray color is used for the change (%) of the specific catalytic activity, white for the change (%) of the diameter of the enzyme molecules. 3
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(unirradiated) sample. When the radiation intensity of 453 J·m−2 was used, the catalytic activity of the enzyme returned to its initial level and retained its value after being exposed with the intensity in the 755–6040 J·m−2 range. After immobilization of bromelain on the high molecular weight chitosan matrix and UV irradiation at intensities of 4530 and 6040 J·m−2, its activity decreased by 21% and 15% respectively. After immobilization of bromelain on the medium molecular weight chitosan matrix, the change in the activity after UV irradiation has not been registered in all the intensity range. No change in the size of the free bromelain molecule under the action of UV irradiation was observed (Fig. 1).
active UV absorption by protein molecules are the residues of aromatic amino acids, first of all tryptophan, much less tyrosine and phenylalanine, and also cystine [35]. Table 2S in Supplementary material presents the data on the content of aromatic amino acids and cysteine in the protease molecules under study. We have not observed any direct correlation between the photosensitivity of the bromelain, ficin and papain molecules, and the number and nature of their chromophores. Fig. 2S of supplementary material shows the results of the amino acid sequences alignment of bromelain, ficin and papain. Color is allocated chromophores for UV irradiation – the residues of tryptophan (W), tyrosine (Y), phenylalanine (F), cysteine (C); raspberry color indicates the active site of enzymes (Cys 26 and His 158 in bromelain, Cys 25 and His 162 in ficin, Cys 25 and His 159 in papain). It is interesting to note that 20 chromophores of UV irradiation at the cysteic proteases under study are parts of conservative area that constitutes 62%, 74% and 57% of the total number of chromophores for bromelain, ficin and papain, respectively. For more detailed analysis of the obtained results we carried out visualization of UV chromophores for bromelain (PDB ID: 1W0Q), ficin (PDB ID: 4YYW), and papain (PDB ID: 9PAP) molecules in relation to their active site (Fig. 3S of supplementary material). For papain and bromelain, the Tyr 170 and Tyr 172 residues are located more closely from their active sites towards His 159 and His 158, respectively. At the same time, ficin has two chromophores – Tyr 177 and Trp 178 – near His 162, that probably causes various UV irradiation effects on the catalytic activity and the molecular size. Fig. 2 shows bonds and interactions between bromelain (PDB ID: 1W0Q), ficin (PDB ID: 4YYW), papain (PDB ID: 9PAP) and chitosan matrix, which arise as a result of the enzyme immobilization. As compared to bromelain and ficin, papain (immobilized on chitosan) demonstrates higher resistance to UV irradiation. This can be explained as follows. Immobilization on chitosan can result in screening some chromophores (amino acid residues) from UV radiation by chitosan molecules. Clearly, the more chromophores are sorbed to the chitosan matrix, the higher is the resistance of the immobilized protein to UVirradiation. After immobilization, ficin has only one chromophore (Trp 184) bound to the chitosan. At the same time, both bromelain and papain have two chromophores immobilized. These chromophore pairs are Tyr 61/Tyr 67 for papain and Trp 180/Phe 29 for bromelain. Given the fact that phenylalanine (quantum yield 0.04) has lower UV absorbance as compared to tyrosine (quantum yield 0.14) and tryptophan (quantum yield 0.13–0.20), one can conclude that the immobilized papain will have two strongly absorbed chromophores screened by chitosan molecules. We have also recorded IR spectra of the high (Fig. 4S of supplementary material) and medium (Fig. 5S of supplementary material) molecular weight chitosans, as well as ficin (Figs. 6S and 7S of supplementary material) bromelain (Figs. 8S and 9S of supplementary material), and papain (Figs. 10S and 11S of supplementary material) immobilized on their matrix, both before and after UV irradiation at 151, 453, 755, 1510, 3020, 4530 and 6040 J·m−2 intensities. IR spectra of ficin, bromelain, papain and both the chitosans are presented in Fig. 12S of supplementary material. Chitosans are characterized by the following peaks in IR spectra: 3600–3100 cm−1 (NeH, OeH), 3100–2800 cm−1 (CeH stretching), 1650–1640 cm−1 (CeN, NeH), 1558–1550 cm−1 (CeN, NeH), 1379–1316 cm−1 (deformation oscillations of OeH and CeH in the
3.2. Photosensitivity of Free and Immobilized Ficin With UV irradiation with intensity from 151 to 1510 J·m−2, the ficin solution did not demonstrated any decrease in its activity. Starting with 3020 J·m−2 and increasing the intensity to 4530 and 6040 J·m−2, a decrease in the catalytic activity of free ficin by 11%, 12% and 17% was registered, respectively. After immobilization of ficin on medium molecular weight chitosan matrix, a tendency to decrease the catalytic activity of the preparation was observed after UV irradiation with 151 J·m−2 intensity. With further irradiation in the intensity range of 453–6040 J·m−2, the enzyme retained its catalytic ability at a relatively constant level. After immobilization of ficin on the high molecular weight chitosan matrix, followed by UV irradiation, the enzyme retained its activity at the initial (i.e. measured before the irradiation) level and no decrease in its catalytic capacity was recorded in the entire intensity range under study. With UV irradiation of free ficin at 3020 J·m−2, some changes of its globule size were detected, possibly due to unfolding of the enzyme molecule (Fig. 1). 3.3. Photosensitivity of Free and Immobilized Papain With UV irradiation of the papain solution at 453 J·m−2 the catalytic activity of the enzyme decreased by 32% compared to the control (non-irradiated) sample. With a further increase of the irradiation intensity, the enzyme maintained its activity at a relatively constant level. After immobilization of papain on the matrix of medium and high molecular weight chitosans, the enzyme maintained its activity at a constant (initial) level after UV irradiation in the range of 151–6040 J·m−2. No change in the size of the papain molecules under the action of UV irradiation was detected in the entire intensity range under study. However, when the samples were irradiated with UV at 755 J·m−2, we detected an increase of the papain globule size. However, this increase was within the confidence interval which also increased under UV irradiation. This may indirectly indicate simultaneous presence of several enzyme forms of different molecular size (Fig. 1). 3.4. Comparative Analysis of the UV Irradiation Effect on Bromelain, Ficin, and Papain Table 1 presents the comparative analysis of the UV irradiation effect on cysteine proteases: bromelain, ficin, and papain. We analyzed the primary structures of bromelain (PDB ID: 1W0Q), ficin (PDB ID: 4YYW), papain (PDB ID: 9PAP) to explain the results. It is known that the chromophore groups responsible for the functionally
Table 1 Comparative analysis of the effect of UV irradiation with intensities in from 151 to 6040 J·m−2 range on cysteine proteases. Enzyme
Catalytic activity
Molecular diameter
Bromelain Ficin Papain
Increases at 151 J·m−2 Decreases at 3020 J·m−2 and higher Decreases at 453 J·m−2 and higher
No changes at any intensity Increases at 3020 J·m−2 Weak (within the confidence interval) increase at 755 J·m−2
4
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Fig. 2. Bonds and interactions between the molecules of cysteine proteases and chitosan matrix.
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Fig. 3. IR spectra of high molecular weight chitosan before and after UV irradiation at 755, 1510, 4530 and 6040 J·m−2.
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pyranose ring), 1200–1000 cm−1 (C–O–C stretching) [33,36–40]. Some bands in IR spectra of high molecular weight chitosan are insensitive to the change of the radiation intensity. These bands are ~1650 cm−1 (primary amides), 1560–1550 cm−1 (deformation vibrations of –N–H and –C–NH2 groups), and 1400–1330 cm−1 (flat and nonplanar deformation vibrations of NH-groups). The intensity of the peaks in the 1000–1070 cm−1 region (corresponding to the stretching vibrations of the –CN– bonds in the primary amino group) increases with exposure to UV irradiation at 755 and 1510 J·m−2, and gradually decreases with further increasing irradiation intensity. At the exposure dose of 755 J·m−2, chitosan molecule probably undergoes some structural changes which are responsible for the peak at 1744 cm−1. This changes get more distinguishable for higher exposure doses resulting in the peaks at 1687 and 1732 cm−1 (4530 J·m−2) and 1744 cm−1 (6040 J·m−2). The shape of IR spectra in the 3000–4000 cm−1 range registered at 755 and 4530 J·m−2 is significantly different from that registered at other exposure doses. These two values of exposure dose are probably correspond to some thresholds in the structural changes of the chitosan molecule (Fig. 3). The changes in IR spectra of the medium molecular weight chitosan under the influence of UV irradiation are more pronounced. Therefore, it undergoes more significant structural modifications than the high molecular chitosan. In particular, there is a decrease in the peak intensity in the 2880–2875 cm−1 region, the peak position being shifted towards larger wave numbers, which is probably due to the modification of certain –CN– and –CH bonds. The maximum of the peak due to the vibrations of the primary amides shifts from 1647 cm−1 (sample without irradiation) to 1651 and 1649 cm−1 at 453 and 755 J·m−2, and moves to 1644–1643 cm−1 as the irradiation intensity increases (shift of the 1643 cm−1 maximum under the influence of UV irradiation was also demonstrated in [38]). The maximum of 897 cm−1 peak shifts gradually to 863 cm−1 with an increase of the irradiation intensity up to 6040 J·m−2, which indicates a change in the frequency of the pyranose cycle's oscillation (Fig. 5S of supplementary material). On the IR spectra of ficin immobilized on both chitosans, the intensity of the amide I band decreases with an increase of the intensity of UV irradiation. When immobilized on medium molecular weight chitosan, the peak of the amide III band shifts towards larger wave numbers (Figs. 6S and 7S of supplementary material). For the bromelain immobilized on high molecular weight chitosan, the maximum of the amide II band shifts from 1555 cm−1 (samples without irradiation) to 1549 cm−1 (after irradiation with intensity of 6040 J·m−2) with an increase of the UV irradiation intensity (Fig. 8S of supplementary material). For the bromelain and papain immobilized on the matrix of medium molecular weight chitosan, no significant changes in the maxima and intensities of the amide I (1630–1690 cm−1), amide II (1520–1560 cm−1), and amide III (1200–1250 cm−1) bands occur after UV irradiation of the samples in the entire range of intensities under study (Fig. 9S and 11S of supplementary material). The increase of irradiation intensity results in the following changes in the IR spectra of papain immobilized on high molecular weight chitosan: the 1649 cm−1 peak (unirradiated sample) is shifted to 1644 cm−1 (after exposure to 6040 J·m−2) with a decrease in the intensity of the amide I band (Fig. 10S of supplementary material). As can be seen from Figs. 6Se11S, the changes in IR spectra of immobilized cysteine proteases practically do not affect the bands due to the protein component of the system: amide I, amide II, amide III. Therefore, it can be concluded that the chitosan matrix acts as a photoprotector for ficin, bromelain and papain immobilized on it.
diameter of the bromelain molecule was observed in the entire range of intensities under study. In the range 151–1510 J·m−2 of UV intensity, the activity of free ficin and its molecules' diameter remains at a constant (initial) level, but both the enzyme activity and its molecular size increase at the UV intensities of 3020, 4530, 6040 J·m−2. The change in the diameter of the molecule and the decrease in the catalytic capacity of the preparation are probably due to unfolding of the protein globule. At the UV intensity of 453 J·m−2, the catalytic activity of papain is decreased and remains at relatively constant level with further irradiation of the preparation. The size of papain molecule shows an increase (however, only within the confidence interval) at 755 J·m−2, but returns to its initial level after irradiation at the intensities in 1510–6040 J·m−2 range. The UV exposure changes more likely the enzymatic activity of free protease molecules than that of the immobilized state. Immobilization leads to an increase in the stability of heterogeneous biocatalysts with respect to UV irradiation in comparison with free enzymes. The chitosan matrix probably plays the role of photoprotector for immobilized ficin, bromelain and papain. The obtained results can be useful in the development of ways to combine the effects of UV radiation, cysteine proteases and chitosan to repair a wound or a burn and to shorten the time of skin healing, as well as when in optimization of the sterilization conditions for the drugs containing ficin, bromelain or papain. Acknowledgements Experimental studies were carried out using the facilities of the Center for Collective Use of Scientific Equipment of Voronezh State University. Formatting Of Funding Sources We acknowledge the support by the Russian Ministry of Science and Higher Education under Grant No. 3.1761.2017/4.6. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jphotobiol.2019.111681. References [1] F. Folgosa, I. Camacho, D. Penas, M. Guilherme, J. Fróis, P.A. Ribeir, P. Tavares, A.S. Pereira, UV irradiation effects on a DNA repair enzyme: conversion of a [4Fe–4S]2+ cluster into a [2Fe–2S]2+, Radiat. Environ. Biophys. 54 (1) (2015) 111–121. [2] D. Hill, J.M. Elwood, D.R. English, Prevention of Skin Cancer, Springer, Dordrecht, 2004. [3] T. Rustemeyer, P. Elsner, S.-M. John, H.I. Maibach, Kanerva’s occupational dermatology, second ed., Springer, Berlin, Heidelberg, 2012. [4] J. Ring, Atopic Dermatitis – Eczema, Springer, Cham, 2016. [5] S.I. Ahmad, Ultraviolet light in human health diseases and environment, Adv. Exp. Med. Biol. 996 (2017) 365. [6] T. Bintsis, E. Litopoulou-Tzanetaki, R.K. Robinson, Existing and potential applications of ultraviolet light in the food industry – a critical review, J. Sci. Food Agric. 80 (2000) 637–645. [7] H. Mohr, U. Gravemann, A. Bayer, T.H. Müller, Sterilization of platelet concentrates at production scale by irradiation with short-wave ultraviolet light, Transfusion 49 (2009) 1956–1963. [8] L. Feijoo-Siota, T.G. Villa, Native and biotechnologically engineered plant proteases with industrial applications, Food Bioprocess Technol. 4 (2011) 1066–1088. [9] M. Esmaeilpour, M.R. Ehsani, M. Aminlari, S. Shekarforoush, E. Hoseini, Antimicrobial activity of peptides derived from enzymatic hydrolysis of goat milk caseins, Comp. Clin. Pathol. 25 (2016) 599–605. [10] P. Alpay, D.A. Uygun, Usage of immobilized papain for enzymatic hydrolysis of proteins, J. Mol. Catal. B Enzym. 111 (2015) 56–63. [11] H.J. Klasen, A review on the non-operative removal of necrotic tissue from burn wounds, Burns 26 (2000) 207–222. [12] B. Thallinger, E.N. Prasetyo, G.S. Nyanhongo, G.M. Guebitz, Antimicrobial enzymes: an emerging strategy to fight microbes and microbial biofilms, Biotechnol. 8
4. Conclusion With a UV irradiation at intensity of 151 J·m−2 we detect an increase of bromelain activity which is reduced as ompared to irradiated samples at the intensities higher than 3020 J·m−2. No change in the 7
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Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Influence of UV radiation on molecular structure and catalytic activity of free and immobilized bromelain, ficin and papain
T
Marina Holyavkaa, , Svetlana Pankovaa, Victoria Korolevaa, Yuliya Vyshkvorkinab, Anatoliy Lukina, Maxim Kondratyevc, Valeriy Artyukhova ⁎
a
Voronezh State University, Universitetskaya sq. 1, Voronezh 394018, Russian Federation Moscow Institute of Physics and Technology (National Research University), Institutskiy per. 9, Dolgoprudny, Moscow Region 141700, Russian Federation c Institute of Cell Biophysics of the Russian Academy of Sciences, Institutskaya st. 3, Puschino, Moscow region 142290, Russian Federation b
ARTICLE INFO
ABSTRACT
Keywords: UV irradiation Adsorption immobilization Bromelain Ficin Papain Chitosan
Our research has shown that the degree of photosensitivity of the cysteine proteases can be arranged in the following order: bromelain → ficin → papain. After the UV irradiation with 151 J·m−2 intensity of a bromelain solution, the enzyme activity has increased. No decrease in the catalytic capacity and the change in the size of the molecule was recorded in the 151–6040 J·m−2 range of irradiation intensities. A decrease in the catalytic capacity of ficin and the increase of its globule size occurred after exposure to a radiation of 3020 J·m−2 intensity. The decrease in papain activity was observed at the UV irradiation intensity of 453 J·m−2, and an increase of the papain globule size was detected at 755 J·m−2. Immobilization on chitosan matrix leads to the increase in the stability of heterogeneous biocatalysts with respect to UV irradiation in comparison with free enzymes. The changes in IR spectra of immobilized cysteine proteases practically do not affect the bands due to the protein component of the system: amide I, amide II, amide III. Therefore, it can be postulated that the chitosan matrix acts as photoprotector for immobilized ficin, bromelain and papain. The obtained results can be helpful for development of drugs based on chitosan and cysteine proteases in combination with phototherapy, as well as for choosing their sterilization conditions.
1. Introduction UV irradiation is one of the generally recognized factors that have a complex impact on organisms at both cellular and molecular levels. Effects from ultraviolet irradiation are initiated by the photochemical absorption of light quanta by molecules of biological significance. The most important of them are nucleic acids, proteins and, in much less degree, some other molecules [1]. UV irradiation can initiate the development of photodermatoses and provoke the development of preand neoplastic processes in human skin. In addition, under the influence of prolonged exposure to UV irradiation, a whole complex of involutive biological processes in various skin layers take place, e.g. photoaging, the clinical manifestation of which is dermatogeliasis [2,3]. Of a particular interest is the study of molecular mechanisms of phototherapy, which is a highly effective option for the treatment of skin diseases, for example, beneficial effects of UV irradiation have
been proven for atopic dermatitis. This method allows to normalize the content of key pro- and anti-inflammatory cytokines in the patients, as UV rays have an impact on the production of immune cells (cytokines), possessing immunosuppressive and anti-inflammatory effects [4]. UV irradiation can influence on activity of various phagocytic cells (neutrophils and dendritic cells), oxidation of blood cell lipids, and various functions of erythrocytes and leukocytes [5]. Its therapeutic effect is based on photochemical reactions that influence on surface receptors and protein (enzymatic) systems of immunocompetent cells. UV irradiation is used to stimulate the components of the immune system and, possible, to exploit the activated immunocytes [3,4]. In addition, the influence of UV irradiation on biomaterials should be investigated systematically, since it is often used for drug sterilization in medical practice [6,7]. Various proteases may act as antimicrobial agents in the treatment of skin lesions. Proteolytic enzymes play a role in several physiological processes: from protein maturation and its degradation to more specific
Corresponding author. E-mail addresses: [email protected] (M. Holyavka), [email protected] (Y. Vyshkvorkina), [email protected] (A. Lukin), [email protected] (V. Artyukhov). ⁎
https://doi.org/10.1016/j.jphotobiol.2019.111681 Received 17 February 2019; Received in revised form 13 October 2019; Accepted 30 October 2019 Available online 01 November 2019 1011-1344/ © 2019 Elsevier B.V. All rights reserved.
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regulation functions. Proteases are the most widely and effectively used enzymes in the food and pharmaceutical industries. They amount about 60% of the global sales of enzymes. Only a few of plant proteases are used industrially. Papain, bromelain and ficin are the most commercially important endopeptidases from plant sources [8,9]. Bromelain (EC 3.4.22.4) is a high molecular weight glycoprotein; its highest content is found in the green pineapple juice. Bromelain is the proteolytic enzyme similar to pepsin and papain, since it breaks down proteins to poly- and oligopeptides. The hydrolysis of these proteins occurs at a wide pH range from 3.0 to 8.0. The molecular weight of bromelain is about 33 kDa; its molecule forms a single polypeptide chain containing 285 amino acid residues. Bromelain has an immunomodulating effect, accelerates tissue repair processes resulted from depolymerization of intercellular structures and modification of vascular permeability. The anti-inflammatory and anti-aggregative effects of bromelain are due to its ability to change the metabolism of arachidonic acid [8,10]. Ficin (EC 3.4.22.3) is a proteolytic enzyme from the latex of the plants genus Ficus. Enzyme refers to cysteine proteases, has a rather wide substrate specificity and shows high activity in the pH range from 6.5 to 9.5. The molecular weight of ficin is 25–26 kDa. The molecule consists of one polypeptide chain. The enzyme preferably hydrolyses the tyrosine and phenylalanine bonds, while neither arginine nor lysine bond splitting has been observed at all. Ficin has antimicrobial activity against Gram-positive and Gram-negative bacteria [8]. Papain (EC 3.4.22.2) is a monothiol cysteine endoprotease. It is active not only in acidic, but also in neutral and alkaline media: pH range from 3.0 to 12.0 (optimum pH 6–7). The molecular weight of papain is 23 kDa. It is a protein consisting of 212 amino acid residues. Papain accelerates the healing of wounds, trophic ulcers, and decubitus by promoting their purification from necrotic masses, accelerates the cleansing of the wound from pathogenic microflora, creates optimal conditions for reparative processes. In biological and biomedical scientific research, papain is widely used in studying the receptor apparatus of cells, the structure of proteins, chemical analysis and purification of various biologically active substances [8,10]. The main disadvantage of the soluble form of proteolytic enzymes is their rapid inactivation due to proteolysis [11,12]. One of the ways to increase the stability of enzymes is their immobilization on polymeric carriers [13,14]. Promising carriers for the immobilization of protein drugs are chitosans and their derivatives, which have antibacterial activity, low allergenicity and relatively low cost [15–21]. In addition, there have been attempts to test chitosan as a carrier for photosensitizers such as porphyrazine. Chitosan effectively prevents photodegradation of their macrocycles [22]. It also forms porous structures and thin films used in cell transplantation and tissue regeneration. In medicine and pharmacy, chitosan is used as a part of bandages, sponges, membranes, artificial leather, contact lenses, funds for the treatment of bone diseases and surgical sutures [23–25]. The properties of chitosan can be modified by adding another polymer [26–30], as well as by the influence of ultraviolet irradiation, mechanical treatment and heating [31–33]. At present, the problem of creating highly effective healing agents with prolonged action for burns and purulent necrotic wounds is not solved. A reason is a long list of requirements imposed to such drugs: non-allergenicity, nontoxicity, biocompatibility and biodegradability, the need to remove the wound exudate, prevention of the wound surface contamination, protection from mechanical damage and drying, possibility for acceleration of skin healing by introducing additional pharmaceuticals substances, ability to maintain the therapeutic effect throughout the storage period. In connection with the emergence and increased reproduction of antibiotic-resistant bacteria, UV irradiation in conjunction with cysteine proteinase therapy should be investigated as an alternative approach to the infection treatment. The complex usage of the UV irradiation, cysteine proteases and chitosan may help to restore the wound or burn faster and to shorten the skin healing time.
Our aim was to study the effects of UV irradiation on photomodulation of bromelain, ficin, papain activity, both free and immobilized on chitosan matrix. 2. Materials and Methods UV irradiation of the free and immobilized cysteine proteases was carried out by their continuous mixing with a magnetic stirrer in a thermostated cell (20 ± 1 °C) using a mercury-quartz lamp of the DRT400 type through a UVS-1 filter with a transmission band of 240–390 nm for 1, 3, 5, 10, 20, 30, 40 min. The intensities of irradiation were 151, 453, 755, 1510, 3020, 4530 and 6040 J·m−2 respectively. The spectral distribution of the DRT-400 lamp's radiation passed through the UVS-1 filter is shown in Fig. 1S of supplementary material. The cysteine proteases – bromelain (B4882), ficin (F4165) and papain (P4762) – were purchased from Sigma-Aldrich, as well as bovine serum albumin (BSA) which served as a substrate for hydrolysis. Two types of acid-soluble chitosan, the medium-molecular weight (200 kDa) and high-molecular weight (350 kDa), were synthesized by Bioprogress Ltd. The UV spectra of the studied systems are shown in Fig. 1S of supplementary material. Immobilization of proteases on the chitosan matrix was carried out by the adsorption method [34]. The catalytic activity of enzymes was determined by the modified Lowry method. Catalytic activity and protein content are presented in Table 1S in Supplementary material. The enzyme amount hydrolizing 1 μM of bovine serum albumin (BSA) per 1 min was taken as a unit of protease activity. The enzymes' molecular size was determined by dynamic light scattering method using the Nano Zetasizer ZS equipment (Malvern Instruments). The back-scattered light from 4 mW He/Ne laser at 632.8 nm wavelength was focused at an angle of 173°. The protein concentration was 1.0–1.5 mg mL−1 in a 0.1 M phosphate buffer at рН 7.4 and a temperature of 25°С. The preparation was passed through a Millipore filter with pores of 0.45 μm in diameter. Registration of infrared spectra of the analyzed samples was carried out on the Bruker Vertex-70 IR-Fourier spectrometer. The spectra were recorded from non-oriented powder samples. Unfortunately, our instrumentation does not allow to measure reduction of the fluorescence lifetime or quantum yield of enzymes after immobilization on chitosan matrix. The amino acid sequences of the studied proteases were aligned using the BioEdit software package. Visualization of chromophores was carried out in the Maestro program. The enzyme structures were prepared for docking according to the standard scheme for Autodock Vina package: the atoms (together with their coordinates) of the solvent, buffer and ligand molecules were removed from the PDB input file. Before carrying out the numerical calculations, a charge was placed on the surface of proteins using MGLTools. The center of the molecule and the parameters of the box («cells») were set manually, ensuring that the whole protease molecule was placed within the computational area of space. The chitosan structure model was drawn in the HyperChem molecular designer; this structure was consistently optimized first in the AMBER force field, and then quantum-chemically in PM3. The ligands in the docking calculations had the maximum conformational freedom: rotation of the functional groups around all single bonds were allowed. Charge arrangement on the chitosan molecule and its protonation/deprotonation was performed automatically in the MGLTools 1.5.6 package. 3. Results and Discussion 3.1. Photosensitivity of Free and Immobilized Bromelain With UV irradiation of bromelain solution at intensity of 151 J·m−2, the enzyme activity was increased by 86% compared to the control 2
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Fig. 1. Effect of UV irradiation on the specific catalytic activity (left), diameter of the enzyme molecules (middle) and their variation in % (right). In the left graph, black color is used for the free enzyme, gray for the enzyme immobilized on medium molecular weight chitosan, white for that immobilized on high molecular weight chitosan. In right graph, gray color is used for the change (%) of the specific catalytic activity, white for the change (%) of the diameter of the enzyme molecules. 3
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(unirradiated) sample. When the radiation intensity of 453 J·m−2 was used, the catalytic activity of the enzyme returned to its initial level and retained its value after being exposed with the intensity in the 755–6040 J·m−2 range. After immobilization of bromelain on the high molecular weight chitosan matrix and UV irradiation at intensities of 4530 and 6040 J·m−2, its activity decreased by 21% and 15% respectively. After immobilization of bromelain on the medium molecular weight chitosan matrix, the change in the activity after UV irradiation has not been registered in all the intensity range. No change in the size of the free bromelain molecule under the action of UV irradiation was observed (Fig. 1).
active UV absorption by protein molecules are the residues of aromatic amino acids, first of all tryptophan, much less tyrosine and phenylalanine, and also cystine [35]. Table 2S in Supplementary material presents the data on the content of aromatic amino acids and cysteine in the protease molecules under study. We have not observed any direct correlation between the photosensitivity of the bromelain, ficin and papain molecules, and the number and nature of their chromophores. Fig. 2S of supplementary material shows the results of the amino acid sequences alignment of bromelain, ficin and papain. Color is allocated chromophores for UV irradiation – the residues of tryptophan (W), tyrosine (Y), phenylalanine (F), cysteine (C); raspberry color indicates the active site of enzymes (Cys 26 and His 158 in bromelain, Cys 25 and His 162 in ficin, Cys 25 and His 159 in papain). It is interesting to note that 20 chromophores of UV irradiation at the cysteic proteases under study are parts of conservative area that constitutes 62%, 74% and 57% of the total number of chromophores for bromelain, ficin and papain, respectively. For more detailed analysis of the obtained results we carried out visualization of UV chromophores for bromelain (PDB ID: 1W0Q), ficin (PDB ID: 4YYW), and papain (PDB ID: 9PAP) molecules in relation to their active site (Fig. 3S of supplementary material). For papain and bromelain, the Tyr 170 and Tyr 172 residues are located more closely from their active sites towards His 159 and His 158, respectively. At the same time, ficin has two chromophores – Tyr 177 and Trp 178 – near His 162, that probably causes various UV irradiation effects on the catalytic activity and the molecular size. Fig. 2 shows bonds and interactions between bromelain (PDB ID: 1W0Q), ficin (PDB ID: 4YYW), papain (PDB ID: 9PAP) and chitosan matrix, which arise as a result of the enzyme immobilization. As compared to bromelain and ficin, papain (immobilized on chitosan) demonstrates higher resistance to UV irradiation. This can be explained as follows. Immobilization on chitosan can result in screening some chromophores (amino acid residues) from UV radiation by chitosan molecules. Clearly, the more chromophores are sorbed to the chitosan matrix, the higher is the resistance of the immobilized protein to UVirradiation. After immobilization, ficin has only one chromophore (Trp 184) bound to the chitosan. At the same time, both bromelain and papain have two chromophores immobilized. These chromophore pairs are Tyr 61/Tyr 67 for papain and Trp 180/Phe 29 for bromelain. Given the fact that phenylalanine (quantum yield 0.04) has lower UV absorbance as compared to tyrosine (quantum yield 0.14) and tryptophan (quantum yield 0.13–0.20), one can conclude that the immobilized papain will have two strongly absorbed chromophores screened by chitosan molecules. We have also recorded IR spectra of the high (Fig. 4S of supplementary material) and medium (Fig. 5S of supplementary material) molecular weight chitosans, as well as ficin (Figs. 6S and 7S of supplementary material) bromelain (Figs. 8S and 9S of supplementary material), and papain (Figs. 10S and 11S of supplementary material) immobilized on their matrix, both before and after UV irradiation at 151, 453, 755, 1510, 3020, 4530 and 6040 J·m−2 intensities. IR spectra of ficin, bromelain, papain and both the chitosans are presented in Fig. 12S of supplementary material. Chitosans are characterized by the following peaks in IR spectra: 3600–3100 cm−1 (NeH, OeH), 3100–2800 cm−1 (CeH stretching), 1650–1640 cm−1 (CeN, NeH), 1558–1550 cm−1 (CeN, NeH), 1379–1316 cm−1 (deformation oscillations of OeH and CeH in the
3.2. Photosensitivity of Free and Immobilized Ficin With UV irradiation with intensity from 151 to 1510 J·m−2, the ficin solution did not demonstrated any decrease in its activity. Starting with 3020 J·m−2 and increasing the intensity to 4530 and 6040 J·m−2, a decrease in the catalytic activity of free ficin by 11%, 12% and 17% was registered, respectively. After immobilization of ficin on medium molecular weight chitosan matrix, a tendency to decrease the catalytic activity of the preparation was observed after UV irradiation with 151 J·m−2 intensity. With further irradiation in the intensity range of 453–6040 J·m−2, the enzyme retained its catalytic ability at a relatively constant level. After immobilization of ficin on the high molecular weight chitosan matrix, followed by UV irradiation, the enzyme retained its activity at the initial (i.e. measured before the irradiation) level and no decrease in its catalytic capacity was recorded in the entire intensity range under study. With UV irradiation of free ficin at 3020 J·m−2, some changes of its globule size were detected, possibly due to unfolding of the enzyme molecule (Fig. 1). 3.3. Photosensitivity of Free and Immobilized Papain With UV irradiation of the papain solution at 453 J·m−2 the catalytic activity of the enzyme decreased by 32% compared to the control (non-irradiated) sample. With a further increase of the irradiation intensity, the enzyme maintained its activity at a relatively constant level. After immobilization of papain on the matrix of medium and high molecular weight chitosans, the enzyme maintained its activity at a constant (initial) level after UV irradiation in the range of 151–6040 J·m−2. No change in the size of the papain molecules under the action of UV irradiation was detected in the entire intensity range under study. However, when the samples were irradiated with UV at 755 J·m−2, we detected an increase of the papain globule size. However, this increase was within the confidence interval which also increased under UV irradiation. This may indirectly indicate simultaneous presence of several enzyme forms of different molecular size (Fig. 1). 3.4. Comparative Analysis of the UV Irradiation Effect on Bromelain, Ficin, and Papain Table 1 presents the comparative analysis of the UV irradiation effect on cysteine proteases: bromelain, ficin, and papain. We analyzed the primary structures of bromelain (PDB ID: 1W0Q), ficin (PDB ID: 4YYW), papain (PDB ID: 9PAP) to explain the results. It is known that the chromophore groups responsible for the functionally
Table 1 Comparative analysis of the effect of UV irradiation with intensities in from 151 to 6040 J·m−2 range on cysteine proteases. Enzyme
Catalytic activity
Molecular diameter
Bromelain Ficin Papain
Increases at 151 J·m−2 Decreases at 3020 J·m−2 and higher Decreases at 453 J·m−2 and higher
No changes at any intensity Increases at 3020 J·m−2 Weak (within the confidence interval) increase at 755 J·m−2
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Fig. 2. Bonds and interactions between the molecules of cysteine proteases and chitosan matrix.
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Fig. 3. IR spectra of high molecular weight chitosan before and after UV irradiation at 755, 1510, 4530 and 6040 J·m−2.
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pyranose ring), 1200–1000 cm−1 (C–O–C stretching) [33,36–40]. Some bands in IR spectra of high molecular weight chitosan are insensitive to the change of the radiation intensity. These bands are ~1650 cm−1 (primary amides), 1560–1550 cm−1 (deformation vibrations of –N–H and –C–NH2 groups), and 1400–1330 cm−1 (flat and nonplanar deformation vibrations of NH-groups). The intensity of the peaks in the 1000–1070 cm−1 region (corresponding to the stretching vibrations of the –CN– bonds in the primary amino group) increases with exposure to UV irradiation at 755 and 1510 J·m−2, and gradually decreases with further increasing irradiation intensity. At the exposure dose of 755 J·m−2, chitosan molecule probably undergoes some structural changes which are responsible for the peak at 1744 cm−1. This changes get more distinguishable for higher exposure doses resulting in the peaks at 1687 and 1732 cm−1 (4530 J·m−2) and 1744 cm−1 (6040 J·m−2). The shape of IR spectra in the 3000–4000 cm−1 range registered at 755 and 4530 J·m−2 is significantly different from that registered at other exposure doses. These two values of exposure dose are probably correspond to some thresholds in the structural changes of the chitosan molecule (Fig. 3). The changes in IR spectra of the medium molecular weight chitosan under the influence of UV irradiation are more pronounced. Therefore, it undergoes more significant structural modifications than the high molecular chitosan. In particular, there is a decrease in the peak intensity in the 2880–2875 cm−1 region, the peak position being shifted towards larger wave numbers, which is probably due to the modification of certain –CN– and –CH bonds. The maximum of the peak due to the vibrations of the primary amides shifts from 1647 cm−1 (sample without irradiation) to 1651 and 1649 cm−1 at 453 and 755 J·m−2, and moves to 1644–1643 cm−1 as the irradiation intensity increases (shift of the 1643 cm−1 maximum under the influence of UV irradiation was also demonstrated in [38]). The maximum of 897 cm−1 peak shifts gradually to 863 cm−1 with an increase of the irradiation intensity up to 6040 J·m−2, which indicates a change in the frequency of the pyranose cycle's oscillation (Fig. 5S of supplementary material). On the IR spectra of ficin immobilized on both chitosans, the intensity of the amide I band decreases with an increase of the intensity of UV irradiation. When immobilized on medium molecular weight chitosan, the peak of the amide III band shifts towards larger wave numbers (Figs. 6S and 7S of supplementary material). For the bromelain immobilized on high molecular weight chitosan, the maximum of the amide II band shifts from 1555 cm−1 (samples without irradiation) to 1549 cm−1 (after irradiation with intensity of 6040 J·m−2) with an increase of the UV irradiation intensity (Fig. 8S of supplementary material). For the bromelain and papain immobilized on the matrix of medium molecular weight chitosan, no significant changes in the maxima and intensities of the amide I (1630–1690 cm−1), amide II (1520–1560 cm−1), and amide III (1200–1250 cm−1) bands occur after UV irradiation of the samples in the entire range of intensities under study (Fig. 9S and 11S of supplementary material). The increase of irradiation intensity results in the following changes in the IR spectra of papain immobilized on high molecular weight chitosan: the 1649 cm−1 peak (unirradiated sample) is shifted to 1644 cm−1 (after exposure to 6040 J·m−2) with a decrease in the intensity of the amide I band (Fig. 10S of supplementary material). As can be seen from Figs. 6Se11S, the changes in IR spectra of immobilized cysteine proteases practically do not affect the bands due to the protein component of the system: amide I, amide II, amide III. Therefore, it can be concluded that the chitosan matrix acts as a photoprotector for ficin, bromelain and papain immobilized on it.
diameter of the bromelain molecule was observed in the entire range of intensities under study. In the range 151–1510 J·m−2 of UV intensity, the activity of free ficin and its molecules' diameter remains at a constant (initial) level, but both the enzyme activity and its molecular size increase at the UV intensities of 3020, 4530, 6040 J·m−2. The change in the diameter of the molecule and the decrease in the catalytic capacity of the preparation are probably due to unfolding of the protein globule. At the UV intensity of 453 J·m−2, the catalytic activity of papain is decreased and remains at relatively constant level with further irradiation of the preparation. The size of papain molecule shows an increase (however, only within the confidence interval) at 755 J·m−2, but returns to its initial level after irradiation at the intensities in 1510–6040 J·m−2 range. The UV exposure changes more likely the enzymatic activity of free protease molecules than that of the immobilized state. Immobilization leads to an increase in the stability of heterogeneous biocatalysts with respect to UV irradiation in comparison with free enzymes. The chitosan matrix probably plays the role of photoprotector for immobilized ficin, bromelain and papain. The obtained results can be useful in the development of ways to combine the effects of UV radiation, cysteine proteases and chitosan to repair a wound or a burn and to shorten the time of skin healing, as well as when in optimization of the sterilization conditions for the drugs containing ficin, bromelain or papain. Acknowledgements Experimental studies were carried out using the facilities of the Center for Collective Use of Scientific Equipment of Voronezh State University. Formatting Of Funding Sources We acknowledge the support by the Russian Ministry of Science and Higher Education under Grant No. 3.1761.2017/4.6. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jphotobiol.2019.111681. References [1] F. Folgosa, I. Camacho, D. Penas, M. Guilherme, J. Fróis, P.A. Ribeir, P. Tavares, A.S. Pereira, UV irradiation effects on a DNA repair enzyme: conversion of a [4Fe–4S]2+ cluster into a [2Fe–2S]2+, Radiat. Environ. Biophys. 54 (1) (2015) 111–121. [2] D. Hill, J.M. Elwood, D.R. English, Prevention of Skin Cancer, Springer, Dordrecht, 2004. [3] T. Rustemeyer, P. Elsner, S.-M. John, H.I. Maibach, Kanerva’s occupational dermatology, second ed., Springer, Berlin, Heidelberg, 2012. [4] J. Ring, Atopic Dermatitis – Eczema, Springer, Cham, 2016. [5] S.I. Ahmad, Ultraviolet light in human health diseases and environment, Adv. Exp. Med. Biol. 996 (2017) 365. [6] T. Bintsis, E. Litopoulou-Tzanetaki, R.K. Robinson, Existing and potential applications of ultraviolet light in the food industry – a critical review, J. Sci. Food Agric. 80 (2000) 637–645. [7] H. Mohr, U. Gravemann, A. Bayer, T.H. Müller, Sterilization of platelet concentrates at production scale by irradiation with short-wave ultraviolet light, Transfusion 49 (2009) 1956–1963. [8] L. Feijoo-Siota, T.G. Villa, Native and biotechnologically engineered plant proteases with industrial applications, Food Bioprocess Technol. 4 (2011) 1066–1088. [9] M. Esmaeilpour, M.R. Ehsani, M. Aminlari, S. Shekarforoush, E. Hoseini, Antimicrobial activity of peptides derived from enzymatic hydrolysis of goat milk caseins, Comp. Clin. Pathol. 25 (2016) 599–605. [10] P. Alpay, D.A. Uygun, Usage of immobilized papain for enzymatic hydrolysis of proteins, J. Mol. Catal. B Enzym. 111 (2015) 56–63. [11] H.J. Klasen, A review on the non-operative removal of necrotic tissue from burn wounds, Burns 26 (2000) 207–222. [12] B. Thallinger, E.N. Prasetyo, G.S. Nyanhongo, G.M. Guebitz, Antimicrobial enzymes: an emerging strategy to fight microbes and microbial biofilms, Biotechnol. 8
4. Conclusion With a UV irradiation at intensity of 151 J·m−2 we detect an increase of bromelain activity which is reduced as ompared to irradiated samples at the intensities higher than 3020 J·m−2. No change in the 7
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