1 Institute of Biomedical Chemistry, Moscow, Russia
2 Foundation of Perspective Technologies and Novations, Moscow, Russia
Flow-based coiled systems, through which a heat transfer fluid (such as glycerol) is pumped, are widely used for thermal stabilization of bioreactors and biosensor cuvettes and cells. Previously, using horseradish peroxidase (HRP) as a model protein, we have demonstrated that the incubation of a protein solution in a flow-based system over coiled pipe with flowing glycerol leads to a change in the adsorption properties of the protein macromolecules. Herein, we have studied the effect of the glycerol flow on the properties of HRP, the solution of which was placed differently: i.e., near either the inflow or the outflow linear sections of the pipe, while the coiled section of the pipe was shielded with a grounded metallic cover. Atomic force microscopy (AFM) has been employed in order to visualize the HRP protein macromolecules adsorbed from its solution onto the mica substrate surface. The quantity of adsorbed protein was estimated based on the AFM data. The enzymatic activity of HRP was estimated by spectrophotometry. We demonstrate that a change in the properties of HRP enzyme was observed after the incubation of its solution near the inflow/outflow linear sections of the pipe with flowing glycerol. Namely, after the incubation of HRP solution near the inflow section, a decrease in the protein adsorption onto mica was observed, but its enzymatic activity remained unchanged in comparison to the control sample. In another case, when the HRP solution was incubated near the outflow section, an increased protein adsorption was observed, while the enzyme exhibited considerably lower activity.
Keywords: horseradish peroxidase; liquid flow; atomic force microscopy; protein aggregation; enzymatic activity; triboelectric effect
In bioreactors and bioanalytical devices, a thermal stabilization of measuring cells and/or reaction vessels is often required to provide optimal process conditions. For this purpose, biosensors and bioreactors can be equipped with flow-based systems with circulating heat transfer fluid , such as water, ethylene glycol, glycerol, etc. Glycerol and glycerol-containing solutions can be employed for thermal stabilization , since their use allows one to vary the process temperature over a broad range from −43.5 °C  to 17 °C . Coiled construction of flow-based systems is a popular technical solution realized in bioanalytical equipment. To achieve optimal heat transfer, the reactor, cuvette, etc. is usually placed within the coil with circulating heat transfer fluid. Previously, we demonstrated that the incubation of a protein solution over the coiled pipe with flowing liquid leads to a change in adsorption properties of the protein macromolecules . We discussed that the flow of a non-aqueous liquid (such as glycerol) generates an electric charge [5,6]. This charge induces an electric field, which, in turn, can affect proteins’ properties.
Herein, we have studied the effect of glycerol flow on the properties of horseradish peroxidase (HRP) model enzyme protein, the solution of which was placed near either the inflow or the outflow linear sections of a flow-based system. In order to eliminate possible interference from the electromagnetic field generated in the coiled section of the system, the coil has been shielded with a grounded metallic cover.
The use of HRP protein in our experiments was justified in our previous papers [5,7,8]. Briefly, this protein was studied in much detail, and this is why it is often employed as a model in studies of enzymes, including peroxidases which play important roles in living systems . In this way, for instance, myeloperoxidase participates in atherogenesis in humans . HRP, in turn, participates in the oxidation of many organic and inorganic compounds by hydrogen peroxide . HRP represents a 40- to 44-kDa [12,13] heme-containing enzyme glycoprotein , containing 18 to 27% carbohydrate residues [13,15,16].
In order to investigate the effect of the glycerol flow on the adsorption properties of HRP, atomic force microscopy (AFM) has been employed. This method allows one to visualize proteins and their complexes at the single-molecule level [17–19]. The use of AFM allowed us to reveal the effect of weak magnetic field  and flow-induced electromagnetic field [5,8] on peroxidase aggregation. In parallel, the enzymatic activity of HRP has been estimated by conventional spectrophotometry.
Many proteins are known to form aggregates, and HRP is one of them . Previously, we demonstrated that a change in the aggregation state of HRP occurs after the exposure of its solution to a weak electromagnetic field . That is, the aggregation state of HRP macromolecules can represent an indicator in order to reveal whether or not an electromagnetic field affects biological macromolecules. Generally, a change in the aggregation state of a protein due to an external influence characterizes alterations in its spatial structure. Such alterations can lead to changes in protein’s functionality and, hence, induce pathologies in an organism as a whole.
Thus, let us, again, emphasize that the present study is aimed at the revelation of the effect of glycerol flow on the properties of HRP (whose solution was placed near either the inflow or the outflow linear section of a coiled polymeric pipe) by AFM and spectrophotometry. In this way, a considerable change in the adsorption properties of this enzyme after incubation of its solution near the inflow section of the pipe has been found. Moreover, both the adsorption properties and the aggregation state of HRP have been revealed to change along with its enzymatic activity after incubation of the enzyme solution near the outflow section of the pipe. This is the main result obtained in our study. The data obtained herein are to be considered in studies of proteins and their complexes with the use of flow-based analytical systems. Our results can find their application in the development of highly sensitive biosensor systems intended for studying structural and functional features of enzymes. Our data can also be useful in studying hemodynamics in human. The latter can be affected by protein aggregation, which can cause such pathologies as cardiovascular  and oncological  diseases.
Ivanov, Y.D.; Pleshakova, T.O.; Shumov, I.D.; Kozlov, A.F.; Ivanova, I.A.; Ershova, M.O.; Tatur, V.Y..; Ziborov, V.S. AFM Study of the Influence of Glycerol Flow on Horseradish Peroxidase Near the In/Out Linear Sections of a Coil. Appl. Sci. 2021, 11, 1723. https://doi.org/10.3390/app11041723