rus
Issue #5/2024
Yu.D.Ivanov, I.D.Shumov, A.N.Ableev, A.F.Kozlov, E.E.Vazhenkova, V.S.Ziborov, A.Yu.Dolgoborodov, O.F.Petrov, S.V.Budnik, R.S.Churyukin, S.V.Novikov, A.M.Tereza, A.I.Archakov
ELECTRON ACCELERATOR FOR INACTIVATION OF HORSERADISH PEROXIDASE ENZYME
ELECTRON ACCELERATOR FOR INACTIVATION OF HORSERADISH PEROXIDASE ENZYME
DOI: https://doi.org/10.22184/1993-8578.2024.17.5.260.266
Nanotechnology manipulates objects with characteristic dimensions of less than 100 nm. In this work, molecules of horseradish peroxidase (HRP) enzyme with dimensions of the order of 5 nm are used as objects, and the device for their inactivation is an electron accelerator, which allows us to obtain electron beam with energy of 9.7 MeV. We demonstrate that upon an irradiation dose of 25 kGy, the activity of the enzyme decreased virtually to zero. The results obtained must be taken into account in the development of methods for sterilization of food and packaging materials for food and medical products.
Nanotechnology manipulates objects with characteristic dimensions of less than 100 nm. In this work, molecules of horseradish peroxidase (HRP) enzyme with dimensions of the order of 5 nm are used as objects, and the device for their inactivation is an electron accelerator, which allows us to obtain electron beam with energy of 9.7 MeV. We demonstrate that upon an irradiation dose of 25 kGy, the activity of the enzyme decreased virtually to zero. The results obtained must be taken into account in the development of methods for sterilization of food and packaging materials for food and medical products.
Теги: electron accelerator enzyme inactivation horseradish peroxidase инактивация фермента пероксидаза хрена электронный ускоритель
INTRODUCTION
Nowadays, an important task of research aimed at the food technology development, biotechnology and biomedicine is the development of methods for inactivation of pathogenic microorganisms (such as bacteria [1] and viral particles [2, 3]) in food [2–6] and sterilisation of medical devices and materials [7]. Published studies have shown the promising use of irradiation of treated media (including packaging materials) with accelerated electrons for the above purposes [5, 6]. The devices developed to date for processing materials with accelerated electrons allow achieving electron energies of the order of 1–10 MeV. In particular, the device TT1000 (Belgium) allows to obtain electron beams with electron energy at the level of 7 MeV [8], and the device of domestic production TEOCORTEX (Russia) – even 10 MeV [9]. Such devices may be used for surface treatment for sterilisation [10]. These units are more convenient to use than chemical sterilisation units, as the electron accelerator-based units are much easier to operate. At the same time, further detailed study of the effect of radiation doses on the treated materials and microorganisms to be inactivated is necessary. In connection with the latter circumstance, it should be noted that processes in living systems, including pathogenic microorganisms, are regulated by enzyme systems [11]. Accordingly, an important area of research is the study of influence of the accelerated electron beams impact on the functioning of enzyme systems.
In our work, we studied inactivation of a model enzyme, horseradish peroxidase, as a result of irradiation of its solution with a beam of accelerated electrons with high energy (~10 MeV). The enzyme HP was chosen as a model object on the basis of its studied [12]: thus, its chemical composition [13] and spatial structure [14] have been characterised. This enzyme is a glycoprotein with a molecular mass of 40–44 kDa [13, 15].
Spectrophotometry was used as a method to determine HP inactivation as a result of exposure of the enzyme solution to an accelerated electron beam, which allows the analysis of enzyme activity after exposure to various physicochemical methods [16–18].
It was shown that an irradiation dose of 25 kGy resulted in an almost complete loss of enzyme activity in the oxidation reaction of its substrate, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), in the presence of hydrogen peroxide (H2O2).
RESEARCH METHODS
Reagents used. Similar to [18], the present work used a HP protein isolated from horseradish (Sigma, Cat. #6782). 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) was also purchased from Sigma (Cat. #A-1888). Hydrogen peroxide (H2O2, h.p.a.), citric acid (o.p.h.) and single-substituted sodium phosphate (Na2HPO4, h.p.a.) were purchased from Reachim (Moscow). 2 mM phosphate-salt buffer in Dulbecco’s modification (PSB-D) was prepared by dissolving a ready-made salt mixture (Pierce, USA) in ultrapure deionised water. All solutions used in the experiments were prepared using ultrapure water (18.2 mOsm · cm) purified using a Simplicity UV unit (Millipore, France).
Electron accelerator experiments. To study the effect of electron beam on the enzyme PC, 1 ml of 10–7 M enzyme solution in 2 mM FSB-D with pH 7.4 was placed in a disposable polypropylene tube with protein solution with a nominal volume of 1.7 ml. The tube was placed in the electron beam zone of the electron accelerator manufactured by "Theocortex". The temperature of the medium was maintained at Tсреды = 20 °C. The radiation dose absorbed by the HP sample irradiated in the electron accelerator was 25 kGy.
Control experiments. Control experiments were performed with a sample of HP kept in an isolated room. A 1 mL control sample of 10–7 M enzyme solution in 2 mM FBS-D buffer with pH 7.4 was also placed in a disposable polypropylene tube containing protein solution with a nominal volume of 1.7 mL, which was placed in an isolation room with a background radiation level allowed by generally accepted health regulations. After that, a control sample of the enzyme solution was also submitted for spectrophotometric analysis.
Spectrophotometric measurements. Spectrophotometric determination of HP enzyme activity was performed according to the method of Sanders et al. [19] as described in our previous publications [16–18]. Specifically, the time-dependent absorbance of 10–9 M solution of HP was recorded at a wavelength of 405 nm for 5 min using a spectrophotometer model 8453 (Agilent Deutschland GmbH, Waldbronn, Germany). The measurements were carried out in quartz cuvettes with an optical path length of 1 cm (Agilent Deutschland GmbH, Waldbronn, Germany). Measurements were performed in phosphate-citrate buffer containing 51 mM Na2HPO4, 24 mM citric acid, pH 5 [19], to which ABTS substrate was also previously added at a concentration of 0.3 mM. For measurements, 30 μl of 0.1 μM (10–7 M) of the tested HP solution was added to a quartz cuvette containing 2.95 ml of the above buffer containing the ABTS substrate and mixed thoroughly. Then 8 µl of 3% (wt./mass) hydrogen peroxide solution was added to the cuvette, and the time dependence of the absorbance of the solution in the cuvette at 405 nm was immediately recorded.
The enzymatic activity of HP in reaction of substrate oxidation by ABTS was calculated from spectrophotometric measurements according to the method of the enzyme and substrate manufacturer (Sigma) [20].
RESULTS AND DISCUSSION
The experiments were carried out in two stages. At the first stage, irradiation of the solution of enzyme HP in the electron accelerator was carried out. The dose of radiation absorbed by this solution was 25 kGy. The control sample of the enzyme solution was kept in an isolated room, excluding its irradiation. At the second stage, enzyme activity was determined in irradiated and control samples of the enzyme solution by spectrophotometry based on the dependence of the product production rate during the reaction of substrate oxidation by ABTS catalysed by HP. For this purpose, the product yield of the reaction was monitored from time for 5 min. During this enzymatic reaction, absorbance of the solution containing the reaction product produced during 5 min at a wavelength of 405 nm and an optical path length of 1 cm was Аконтр = 2.0 and Аоблуч = 0.06 for the control and 25 kGy irradiated enzyme samples, respectively. Accordingly, the activity of the enzyme HP relative to its substrate ABTS in the control and irradiated samples was 111.67 units/ml and 3.35 units/ml, having decreased as a result of irradiation by 33 times.
The data obtained by us are in agreement with those obtained earlier by Liu et al. [21]. These authors studied the effect of electron beam irradiation on the activity of enzymes of cyanobacteria Microcystis aeruginosa and found a ~ 10-fold decrease in the activity of Microcystis aeruginosa peroxidase after irradiation with an electron beam energy of only 1 MeV at irradiation doses of even 5 kGy. In our case, the beam energy was 10 times higher and the irradiation dose was 5 times higher than in the experiments reported by Liu et al. [21], which led to even stronger deactivation of the peroxidase enzyme. In our work, we carried out studies on the effect of irradiation with an electron beam of ~ 10 MeV energy on the enzyme HP. The spectrophotometric method was used to control the activity of the enzyme HP. Based on spectrophotometric data it was obtained that the enzyme activity without irradiation was 33 times higher than after irradiation with a dose of 25 kGy. It is known that enzyme activity is related to its spatial structure, including the structure of its active cite [11]. Accordingly, the presented data mean that at an irradiation dose of about 25 kGy, destruction of the enzyme structure, including its active centre, is observed.
CONCLUSIONS
The effect of accelerated electrons with an energy of ~10 MeV on the HP enzyme was studied. It was obtained that at such an irradiation dose almost complete inactivation of the enzyme was observed, indicating the destruction of its structure. The results are important for correct assessment of accelerated electrons influence on enzymes during sterilisation of foodstuffs and other materials in installations based on electron accelerators. Also, the results of our work should be taken into account to create safe working conditions for personnel.
ACKNOWLEDGMENTS
Spectrophotometric measurements were performed within the framework of the Programme of Fundamental Scientific Research in the Russian Federation for a Long-term Period (2021–2030) (№122030100168-2). The enzyme irradiation in an electron accelerator was supported by the Ministry of Science and Higher Education of the Russian Federation (Agreement No. 075-00270-24-00).
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Nowadays, an important task of research aimed at the food technology development, biotechnology and biomedicine is the development of methods for inactivation of pathogenic microorganisms (such as bacteria [1] and viral particles [2, 3]) in food [2–6] and sterilisation of medical devices and materials [7]. Published studies have shown the promising use of irradiation of treated media (including packaging materials) with accelerated electrons for the above purposes [5, 6]. The devices developed to date for processing materials with accelerated electrons allow achieving electron energies of the order of 1–10 MeV. In particular, the device TT1000 (Belgium) allows to obtain electron beams with electron energy at the level of 7 MeV [8], and the device of domestic production TEOCORTEX (Russia) – even 10 MeV [9]. Such devices may be used for surface treatment for sterilisation [10]. These units are more convenient to use than chemical sterilisation units, as the electron accelerator-based units are much easier to operate. At the same time, further detailed study of the effect of radiation doses on the treated materials and microorganisms to be inactivated is necessary. In connection with the latter circumstance, it should be noted that processes in living systems, including pathogenic microorganisms, are regulated by enzyme systems [11]. Accordingly, an important area of research is the study of influence of the accelerated electron beams impact on the functioning of enzyme systems.
In our work, we studied inactivation of a model enzyme, horseradish peroxidase, as a result of irradiation of its solution with a beam of accelerated electrons with high energy (~10 MeV). The enzyme HP was chosen as a model object on the basis of its studied [12]: thus, its chemical composition [13] and spatial structure [14] have been characterised. This enzyme is a glycoprotein with a molecular mass of 40–44 kDa [13, 15].
Spectrophotometry was used as a method to determine HP inactivation as a result of exposure of the enzyme solution to an accelerated electron beam, which allows the analysis of enzyme activity after exposure to various physicochemical methods [16–18].
It was shown that an irradiation dose of 25 kGy resulted in an almost complete loss of enzyme activity in the oxidation reaction of its substrate, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), in the presence of hydrogen peroxide (H2O2).
RESEARCH METHODS
Reagents used. Similar to [18], the present work used a HP protein isolated from horseradish (Sigma, Cat. #6782). 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) was also purchased from Sigma (Cat. #A-1888). Hydrogen peroxide (H2O2, h.p.a.), citric acid (o.p.h.) and single-substituted sodium phosphate (Na2HPO4, h.p.a.) were purchased from Reachim (Moscow). 2 mM phosphate-salt buffer in Dulbecco’s modification (PSB-D) was prepared by dissolving a ready-made salt mixture (Pierce, USA) in ultrapure deionised water. All solutions used in the experiments were prepared using ultrapure water (18.2 mOsm · cm) purified using a Simplicity UV unit (Millipore, France).
Electron accelerator experiments. To study the effect of electron beam on the enzyme PC, 1 ml of 10–7 M enzyme solution in 2 mM FSB-D with pH 7.4 was placed in a disposable polypropylene tube with protein solution with a nominal volume of 1.7 ml. The tube was placed in the electron beam zone of the electron accelerator manufactured by "Theocortex". The temperature of the medium was maintained at Tсреды = 20 °C. The radiation dose absorbed by the HP sample irradiated in the electron accelerator was 25 kGy.
Control experiments. Control experiments were performed with a sample of HP kept in an isolated room. A 1 mL control sample of 10–7 M enzyme solution in 2 mM FBS-D buffer with pH 7.4 was also placed in a disposable polypropylene tube containing protein solution with a nominal volume of 1.7 mL, which was placed in an isolation room with a background radiation level allowed by generally accepted health regulations. After that, a control sample of the enzyme solution was also submitted for spectrophotometric analysis.
Spectrophotometric measurements. Spectrophotometric determination of HP enzyme activity was performed according to the method of Sanders et al. [19] as described in our previous publications [16–18]. Specifically, the time-dependent absorbance of 10–9 M solution of HP was recorded at a wavelength of 405 nm for 5 min using a spectrophotometer model 8453 (Agilent Deutschland GmbH, Waldbronn, Germany). The measurements were carried out in quartz cuvettes with an optical path length of 1 cm (Agilent Deutschland GmbH, Waldbronn, Germany). Measurements were performed in phosphate-citrate buffer containing 51 mM Na2HPO4, 24 mM citric acid, pH 5 [19], to which ABTS substrate was also previously added at a concentration of 0.3 mM. For measurements, 30 μl of 0.1 μM (10–7 M) of the tested HP solution was added to a quartz cuvette containing 2.95 ml of the above buffer containing the ABTS substrate and mixed thoroughly. Then 8 µl of 3% (wt./mass) hydrogen peroxide solution was added to the cuvette, and the time dependence of the absorbance of the solution in the cuvette at 405 nm was immediately recorded.
The enzymatic activity of HP in reaction of substrate oxidation by ABTS was calculated from spectrophotometric measurements according to the method of the enzyme and substrate manufacturer (Sigma) [20].
RESULTS AND DISCUSSION
The experiments were carried out in two stages. At the first stage, irradiation of the solution of enzyme HP in the electron accelerator was carried out. The dose of radiation absorbed by this solution was 25 kGy. The control sample of the enzyme solution was kept in an isolated room, excluding its irradiation. At the second stage, enzyme activity was determined in irradiated and control samples of the enzyme solution by spectrophotometry based on the dependence of the product production rate during the reaction of substrate oxidation by ABTS catalysed by HP. For this purpose, the product yield of the reaction was monitored from time for 5 min. During this enzymatic reaction, absorbance of the solution containing the reaction product produced during 5 min at a wavelength of 405 nm and an optical path length of 1 cm was Аконтр = 2.0 and Аоблуч = 0.06 for the control and 25 kGy irradiated enzyme samples, respectively. Accordingly, the activity of the enzyme HP relative to its substrate ABTS in the control and irradiated samples was 111.67 units/ml and 3.35 units/ml, having decreased as a result of irradiation by 33 times.
The data obtained by us are in agreement with those obtained earlier by Liu et al. [21]. These authors studied the effect of electron beam irradiation on the activity of enzymes of cyanobacteria Microcystis aeruginosa and found a ~ 10-fold decrease in the activity of Microcystis aeruginosa peroxidase after irradiation with an electron beam energy of only 1 MeV at irradiation doses of even 5 kGy. In our case, the beam energy was 10 times higher and the irradiation dose was 5 times higher than in the experiments reported by Liu et al. [21], which led to even stronger deactivation of the peroxidase enzyme. In our work, we carried out studies on the effect of irradiation with an electron beam of ~ 10 MeV energy on the enzyme HP. The spectrophotometric method was used to control the activity of the enzyme HP. Based on spectrophotometric data it was obtained that the enzyme activity without irradiation was 33 times higher than after irradiation with a dose of 25 kGy. It is known that enzyme activity is related to its spatial structure, including the structure of its active cite [11]. Accordingly, the presented data mean that at an irradiation dose of about 25 kGy, destruction of the enzyme structure, including its active centre, is observed.
CONCLUSIONS
The effect of accelerated electrons with an energy of ~10 MeV on the HP enzyme was studied. It was obtained that at such an irradiation dose almost complete inactivation of the enzyme was observed, indicating the destruction of its structure. The results are important for correct assessment of accelerated electrons influence on enzymes during sterilisation of foodstuffs and other materials in installations based on electron accelerators. Also, the results of our work should be taken into account to create safe working conditions for personnel.
ACKNOWLEDGMENTS
Spectrophotometric measurements were performed within the framework of the Programme of Fundamental Scientific Research in the Russian Federation for a Long-term Period (2021–2030) (№122030100168-2). The enzyme irradiation in an electron accelerator was supported by the Ministry of Science and Higher Education of the Russian Federation (Agreement No. 075-00270-24-00).
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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