1. Institute of Biomedical Chemistry, Moscow

2. Joint Institute for High Temperatures of the Russian Academy of Sciences, Moscow

3. Foundation of Perspective Technologies and Novations, Moscow

4. Moscow State Academy of Veterinary Medicine and Biotechnology Named after Skryabin, Moscow

5. Faculty of Computational Mathematics and Cybernetics, Moscow State University, Moscow

Glycerol has found its applications as a heat-transfer fluid in heat exchangers, and as a component of functional liquids in biosensor analysis. Flowing non-aqueous fluids are known to be able to induce electromagnetic fields due to the triboelectric effect. These triboelectrically generated electromagnetic fields can affect biological macromolecules. Horseradish peroxidase (HRP) is widely employed as a convenient model object for studying how external electric, magnetic, and electromagnetic fields affect enzymes. Herein, we have studied whether the flow of glycerol in a ground-shielded cylindrical coil affects the HRP enzyme incubated at a 2 cm distance near the coil’s side. Atomic force microscopy (AFM) has been employed in order to study the effect of glycerol flow on HRP at the nanoscale. An increased aggregation of HRP on mica has been observed after the incubation of the enzyme near the coil. Moreover, the enzymatic activity of HRP has also been affected. The results reported that their application can be found in biotechnology, food technology and life sciences applications, considering the development of triboelectric generators, enzyme-based biosensors and bioreactors with surface-immobilized enzymes. Our work can also be of interest for scientists studying triboelectric phenomena, representing one more step toward understanding the mechanism of the indirect action of the flow of a dielectric liquid on biological macromolecules.

Keywords: horseradish peroxidase; glycerol; enzyme aggregation; enzymatic activity; liquid flow

Glycerol, a trihydric alcohol, is employed as a heat transfer fluid [1], representing a non-toxic alternative to widely used ethylene glycol [2]. Furthermore, glycerol finds its application in flow-based biosensors as a component of specialized buffer solutions [3]. The abovementioned applications can be used for the organization of a fluid flow.

Regarding biosensor systems, biological macromolecules under study are either introduced directly into the flow or immobilized on the sensor surface, which is in direct physical contact with the fluid flow [3,4,5,6,7]. In this situation, the fluid flow is directly interacting with the biological macromolecules. Regarding bovine serum albumin (BSA), Dobson et al. demonstrated that the direct action of a fluid flow on biological macromolecules can induce their aggregation [8]. It should be emphasized that the fluid flow can also act on biological macromolecules indirectly. In other words, the fluid flow can affect biological macromolecules even when there is no direct mechanical contact between the molecules and the flow. Below, we consider one of the possible mechanisms of the indirect action of the fluid flow on enzymes.

The flow of both aqueous [9,10,11,12,13,14,15] and non-aqueous [16,17,18,19,20,21] fluids can well cause the triboelectric generation of electric charge [9,10,11,12,13,14,15,16,17,18,19,20,21]. This widely employed phenomenon is the development of the so-called triboelectric generators [11,12,13,14,18]. It should be emphasized that, despite the fact that aqueous fluids are used for this purpose, in the majority of cases [11,12,13,14], the successful development of oil-employing triboelectric devices was recently reported [18]. Yoo et al. have found that, in the case of glycerol, the triboelectric generation of charge is very significant in comparison with the majority of other liquids tested [21]. In this context, it should be borne in mind that triboelectrically induced fields can influence biological macromolecules [15,16,17], exhibiting an indirect action of a fluid flow on these macromolecules. Additional attention should also be paid in the case of the triboelectric effect of fluid flow through pipes [9,21], including pipe coils, which are used as heat exchangers in bioreactors [22]. Heat exchangers are key components of bioreactors operating with enzymes [23,24]. Since glycerol, which was found to be triboelectrically active [21], is often used as a heat transfer agent [1], further research is required in order to investigate the possible effects of its flow on biological molecules, including enzymes. And this is the aim of our study.

The effect of an external triboelectrically induced field on biological macromolecules is often manifested in the form of the aggregation of these macromolecules under the field action [15,16,17]. Atomic force microscopy (AFM) represents a powerful method, which allows one to investigate the aggregation of biological macromolecules [25,26,27], including enzymes [15,16,17,28,29,30,31,32,33,34] with high (up to single-molecule) resolution. This feature of AFM allows one to even reveal subtle effects, which are indistinguishable by macroscopic methods such as spectrophotometry [29].

AFM is widely employed for studying the aggregation state of various enzymes. In [31], AFM has allowed to determine the ratio between monomers and oligomers of cytochrome CYP102A1, whose oligomers were reported to have a higher enzymatic activity than its monomers [35]. Namely, with the use of an AFM probe with typical (10 nm) tip curvature radius, this ratio was found to be 0.5:0.5. Moreover, the use of a supersharp AFM probe with tip curvature radius of 2 nm has allowed the determination of the ratio between different oligomers of this enzyme (dimers/trimers/tetramers = 0.3:0.1:0.1). This example clearly illustrates the excellent suitability of AFM for enzyme aggregation studies. Furthermore, Baron et al. [32] discussed the influence of isopropanol on enzyme–enzyme and enzyme–surface interactions on the adsorption of lipase (isolated from Bacillus megaterium CCOC P2637) on polypropylene and silicon substrates. These authors have demonstrated that the addition of isopropanol to a buffered aqueous solution of the lipase promotes its disaggregation, forcing its adsorption onto hydrophilic surfaces. With the example of glutamate dehydrogenase, Blasi et al. [33] and Zhang and Tan [34] demonstrated the applications of AFM for the characterization of enzyme-functionalized surfaces.

Horseradish peroxidase (HRP) represents a ~44 kDa [36] enzyme glycoprotein, whose structure is stabilized by carbohydrate chains [37,38]. Since this enzyme is comprehensively characterized in the literature [39], many authors have used it as a model object for studying the effect of electric [40], magnetic [41,42,43,44] and electromagnetic [15,16,17,45,46,47,48] fields on enzymes. Previously, we reported the successful use of AFM for the revelation of HRP aggregation under the influence of flow-induced electromagnetic fields [15,16,17]. Sun et al. [43,44] employed AFM for the investigation of the influence of alternating magnetic fields on HRP adsorption onto mica, and revealed that the action of the field on the enzyme forces it to form complex extended structures on the substrate surface. Wasak et al. [41] studied the effect of low-frequency rotating magnetic fields on HRP. These authors have found an increase in the enzymatic activity of HRP against o-dianisidine at 1 Hz and 20 Hz magnetic field frequencies (at which the activity increased by 8% and 12%, respectively), while the action of the field of other frequencies studied (2; 5; 10; 30; 40 and 50 Hz) were found to deactivate the enzyme. Emamdadi et al. [42] observed a very significant enhancement in the enzymatic activity of HRP against pyrogallol after its 30 min incubation in a 52 mT static magnetic field. Regarding radio frequency electromagnetic fields, Fortune et al. [45] have observed no nonthermal effect on HRP after the action of either a 13.56 MHz, 915 MHz or 2.45 GHz frequency. Of note, Yao et al. [46] have recently reported very interesting results regarding the activation of HRP by its radio frequency (27.12 MHz) heating at 50 °C.

Our present research is aimed at studying the indirect effect of glycerol flow through a cylindrical coil heat exchanger on HRP. With this purpose, we have employed the well-established approach based on AFM and spectrophotometry [15,17,28] for studying the effect of glycerol flow in a shielded coil on HRP. While AFM has allowed us to reveal an increase in the aggregation of HRP on the surface of mica substrates, an increase in its enzymatic activity against its substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in solution has also been observed.

The data reported can be of interest for scientists studying the interaction of biological macromolecules with electromagnetic fields. It is to be emphasized that HRP has found numerous applications in biotechnology as a catalyst [49], and in diagnostics as a reporter enzyme [50]. Accordingly, our results can also find application in the development of water purification systems [51,52], food treatment methods [46] and biomarker detection systems [53], considering the development of enzyme-based biosensors [53] and bioreactors with surface-immobilized enzymes [52].

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Ivanov, Yuri D., Ivan D. Shumov, Andrey F. Kozlov, Maria O. Ershova, Anastasia A. Valueva, Irina A. Ivanova, Vadim Y. Tatur, Andrei A. Lukyanitsa, Nina D. Ivanova, and Vadim S. Ziborov. 2023. "Glycerol Flow through a Shielded Coil Induces Aggregation and Activity Enhancement of Horseradish Peroxidase" Applied Sciences 13, no. 13: 7516. https://doi.org/10.3390/app13137516