1 Institute of Biomedical Chemistry, Moscow, Russia
2 Joint Institute for High Temperatures of the Russian Academy of Sciences, Moscow, Russia
3 Foundation of Perspective Technologies and Novations, Moscow, Russia
4 Skryabin Moscow State Academy of Veterinary Medicine and Biotechnology, Moscow, Russia
5 Bruker Ltd., Moscow, Russia
External electromagnetic fields are known to be able to concentrate inside the construction elements of biosensors and bioreactors owing to reflection from their surface. This can lead to changes in the structure of biopolymers (such as proteins), incubated inside these elements, thus influencing their functional properties. Our present study concerned the revelation of the effect of spherical elements, commonly employed in biosensors and bioreactors, on the physicochemical properties of proteins with the example of the horseradish peroxidase (HRP) enzyme. In our experiments, a solution of HRP was incubated within a 30 cm-diameter titanium half-sphere, which was used as a model construction element. Atomic force microscopy (AFM) was employed for the single-molecule visualization of the HRP macromolecules, adsorbed from the test solution onto mica substrates in order to find out whether the incubation of the test HRP solution within the half-sphere influenced the HRP aggregation state. Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was employed in order to reveal whether the incubation of HRP solution within the half-sphere led to any changes in its secondary structure. In parallel, spectrophotometry-based estimation of the HRP enzymatic activity was performed in order to find out if the HRP active site was affected by the electromagnetic field under the conditions of our experiments. We revealed an increased aggregation of HRP after the incubation of its solution within the half-sphere in comparison with the control sample incubated far outside the half-sphere. ATR-FTIR allowed us to reveal alterations in HRP’s secondary structure. Such changes in the protein structure did not affect its active site, as was confirmed by spectrophotometry. The effect of spherical elements on a protein solution should be taken into account in the development of the optimized design of biosensors and bioreactors, intended for performing processes involving proteins in biomedicine and biotechnology, including highly sensitive biosensors intended for the diagnosis of socially significant diseases in humans (including oncology, cardiovascular diseases, etc.) at early stages.
Keywords: atomic force microscopy; horseradish peroxidase; protein aggregation; electromagnetic field; bioreactor
Proteins, representing polymers of amino acids, are among the main types of biopolymers, playing various vital functions in living organisms . Proteins can function as single-macromolecule structures or in the form of various complexes, including (but not limited to) protein oligomers and protein-protein, protein–nucleic acid, and protein–small molecule complexes . In modern life, electromagnetic fields are widely employed, influencing living organisms. The functionality of protein systems (including enzymatic ones) can be altered under the action of magnetic  and electromagnetic fields [3–10]. In this way, in previous studies, we demonstrated that electric fields, triboelectrically induced by liquid flow through polymeric pipes of thermal stabilization coils, influence the adsorbability of the horseradish peroxidase (HRP) enzyme protein onto mica substrates [5–7]. Moreover, under certain conditions, its enzymatic activity can also be affected by the flow-induced field . A 40 min exposure to an external ultra-weak (10-12 W/cm2) 2.3 GHz knotted electromagnetic field was also shown to have an effect on the HRP aggregation state upon its adsorption onto mica . Lopes et al.  found that 2450 MHz  microwave radiation can cause a significant (up to >80%) loss in the HRP enzymatic activity after a 0.5 h treatment at 600C and 60W microwave power. Hamedi et al. demonstrated partial unfolding of adult hemoglobin (HbA) after exposure to a 940 MHz circularly polarized electromagnetic field . As regards HRP, a 52 mT static magnetic field was also shown to impact its enzymatic activity and optimum pH by inducing changes in its structure .
As regards proteins, alterations in their functionality can manifest themselves not only as direct changes in functional activity [2,3,9], but also in the form of changes in adsorbability [5–7,10] onto functional surfaces of biosensors and bioreactors. The latter is of importance for biotechnology applications, where bioreactors with surface-immobilized enzymes are widely employed [12,13].
Spherical elements are commonly used in the construction of bioreactors, including bioreactor bottoms [14,15]. Previous theoretical studies demonstrated the ability of large pyramidal objects to concentrate weak electromagnetic radiation near their surface and within their volume, which is attributed to resonance phenomena . These phenomena are supposed to be the very cause of the effect of incubation near a pyramidal structure on HRP adsorbability from aqueous solutions onto mica . In this connection, it should be noted that electromagnetic field-induced protein aggregation can lead to the occurrence of pathologies in the body, for instance by influencing the rheological properties of blood [17,18]. It should be emphasized that the effects on blood rheology were observed in the case of electromagnetic fields of commercial 50 Hz mains frequency , commonly employed in both industry and everyday life. Since reaction vessels with spherical bottoms are widely employed in industry, it is particularly important to study the influence of external electromagnetic fields of commercial frequency, concentrated by spherical construction elements of bioreactors, on protein systems.
The horseradish peroxidase (HRP) enzyme protein is widely employed in biotechnology . The wide use in both research and industrial applications makes it important to study the influence of electromagnetic fields of commercial frequency on its properties. Moreover, the availability of detailed and comprehensive information about its structure and physicochemical properties makes it easier to interpret the effects observed in the experiments, and this is why HRP is a useful model object in studying the external impacts on the properties of proteins. Structurally, HRP represents a 40–44 kDa [19,20] heme-containing enzyme glycoprotein , which contains 18–27% structure-stabilizing carbohydrate residues [20,22]. In micromolar aqueous solutions, HRP is prone to aggregation , while being presented in monomeric form at ultra-low concentrations .
Atomic force microscopy (AFM) represents a high-resolution method commonly employed in studying single-polymer macromolecules at the nanoscale . Studying biopolymers such as proteins [24,26–29] and nucleic acids [30–34] is one of the main directions in the development of AFM applications . Owing to its extremely high (~0.1 nm) height resolution, AFM allows one to visualize single biological macromolecules and their complexes. Moreover, AFM allows one to determine the physicochemical properties of proteins at the single-molecule level, such as Young’s modulus , aggregation state [4,27], and enzymatic activity [29,36]. Since AFM allows one to study single-polymer macromolecules, operating at the nanoscale, it allows one to reveal subtle effects , which are indistinguishable by macroscopic methods . In our previous studies, the Polymers 2021, 13, 1601 3 of 13 AFM-based approach allowed us to reveal the effects of external electromagnetic fields on the adsorption properties and aggregation state of HRP [4–7,10].
Our present study concerned the atomic force microscopy (AFM)-based revelation of the effect of a half-spherical element, whose shape was similar to the commonly used construction elements of bioreactor vessels, on the properties of the HRP model enzyme protein. The study was performed under common laboratory conditions, and the experimental setup, including the spherical element, was devoid of any electromagnetic shielding. We emphasize that in our experiments reported herein, external electromagnetic radiation was not generated intentionally by using specialized generators, as opposed to our previously reported experiments . In addition, the attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) method was employed in order to reveal whether or not the incubation of the HRP solution within the half-sphere led to any changes in its secondary structure. Moreover, spectrophotometry-based estimation of the HRP’s enzymatic activity was performed in order to find out whether or not the HRP active site was affected by the electromagnetic field under the conditions of our experiments. In our experiments reported herein, an increased aggregation of HRP after the incubation of its aqueous solution inside a titanium half-sphere was observed. The ATR-FTIR data obtained indicated alterations in HRP’s secondary structure after the incubation of its solution in the center of the half-sphere. At the same time, spectrophotometry results indicated no change in HRP’s enzymatic activity. These results indicated the importance of further studying how electromagnetic fields of commercial (50 Hz) frequency, generated by various equipment in both industry and everyday life, can affect living systems. This is required in order to develop safety standards regulating the application of electromagnetic field-inducing equipment in industry, where technological vessels with spherical construction elements are commonly employed. This is why the development of novel highly sensitive biosensor systems, which allow one to perform measurements at the single-molecule level, represents a crucial problem in biomedical research. The development of such biosensors will help to better understand the influence of external electromagnetic fields on humans. Moreover, the application of such systems will allow us to solve a number of important problems in biomedicine, including the early diagnosis of somatic and infectious diseases (such as cancer, cardiovascular diseases, hepatitis, and other viral infections) in humans.
Ivanov, Y.D.; Tatur, V.Y.; Pleshakova, T.O.; Shumov, I.D.; Kozlov, A.F.; Valueva, A.A.; Ivanova, I.A.; Ershova, M.O.; Ivanova, N.D.; Repnikov, V.V.; Stepanov, I.N.; Ziborov, V.S. Effect of Spherical Elements of Biosensors and Bioreactors on the Physicochemical Properties of a Peroxidase Protein. Polymers 2021, 13, 1601. https://doi.org/10.3390/polym13101601