AFM Imaging of Protein Aggregation in Studying the Impact of Knotted Electromagnetic Field on A Peroxidase

Abstract

The phenomenon of knotted electromagnetic field (KEMF) is now actively studied, as such fields are characterized by a nontrivial topology. The research in this field is mainly aimed at technical applications-for instance, the development of efficient communication systems. Until present, however, the influence of KEMF on biological objects (including enzyme systems) was not considered. Herein, we have studied the influence of KEMF on the aggregation and enzymatic activity of a protein with the example of horseradish peroxidase (HRP). The test HRP solution was irradiated in KEMF (the radiation power density was 10 −12 W/cm 2 at 2.3 GHz frequency) for 40 min. After the irradiation, the aggregation of HRP was examined by atomic force microscopy (AFM) at the single-molecule level. The enzymatic activity was monitored by conventional spectrophotometry. It has been demonstrated that an increased aggregation of HRP, adsorbed on the AFM substrate surface, was observed after irradiation of the protein sample in KEMF with low (10 −12 W/cm 2) radiation power density; at the same time, the enzymatic activity remained unchanged. the results obtained herein can be used in the development of models describing the interaction of enzymes with electromagnetic field. The obtained data can also be of importance considering possible pathological factors that can take place upon the influence of KEMF on biological objects-for instance, changes in hemodynamics due to increased protein aggregation are possible; the functionality of protein complexes can also be affected by aggregation of their protein subunits. These effects should also be taken into account in the development of novel highly sensitive systems for human serological diagnostics of breast cancer, prostate cancer, brain cancer and other oncological pathologies, and for diagnostics of diseases in animals, and crops. It is known that electromagnetic radiation of various intensity can have different influence on human body. Electromagnetic fields can have various topology, such as transverse and knotted one (knotted electromagnetic field, KEMF) 1,2. The simplest and most common electromagnetic waves are transverse ones, and, to date, their effects have been widely studied in various frequency and intensity ranges. As regards biomedical applications, microwave radiation is interesting in that, depending on its intensity, it is employed in biological research, and in both medical diagnostics and therapy. In this way, upon exposure of biological tissues to high-intensity radiation (~90 W/cm 2), their temperature increases to ~90 °С, and denaturation of biological objects is observed. It was shown that, under such conditions, partial loss of functional activity of proteins (for instance, peroxidase) is observed 3. At lower radiation intensity (10 μW/cm 2 4), both positive therapeutic effects (which usually take place owing to local heating 5) and negative effects are observed. Here, it should be noted that studies on the application of non-thermal effects of low-power microwave r...

AFM and FTIR Investigation of the Effect of Water Flow on Horseradish Peroxidase

“…This fact indicates that the flow-induced field only impacts the spatial structures of the protein globule near its surface, while not affecting its active site. The effects of HRP aggregation in the presence of electromagnetic fields were previously reported in our recent studies [ 8 , 35 , 36 ] and by Sun et al [ 37 , 38 ].…”

“…The phenomenon observed is also to be taken into account in the development of biosensor systems. This is particularly important in highly sensitive biosensors, intended for the diagnosis of various types of cancer (such as brain cancer, prostate cancer, breast cancer, and ovarian cancer) in humans [ 35 ]. Since the peroxidase enzyme changes its adsorbability under the influence of an electromagnetic field, then the influence of electromagnetic radiation on inflammatory processes is also possible.…”

“…The AFM experiments were carried out using the direct surface adsorption method [ 8 , 15 , 35 , 36 ], similar to [ 8 ]. Briefly, muscovite mica sheets (SPI, West Chester, PA, USA) were used as AFM substrates.…”

“…HRP activity was estimated following the technique reported by Sanders et al [ 48 ], employing ABTS as a reducing substrate—similar to our previous papers [ 8 , 35 ]. The rate of change in solution absorbance at 405 nm was monitored with an Agilent 8453 UV-visible spectrophotometer (Agilent Technologies Deutschland GmbH, Waldbronn, Germany).…”

AFM Study of the Influence of Glycerol Flow on Horseradish Peroxidase near the in/out Linear Sections of a Coil

“…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 [9].…”