## 4 Discussion  
As was noted in the introduction, in the development of novel diagnostics intended for the registration of protein aggregation, and medicinal agents, aimed at the regulation of the aggregation process, it is important to study the factors, which influence the formation of protein aggregates. In the present research, the influence of pulsed electromagnetic and pressure fields with a very short rise-time of the front on the HRP enzyme protein has been investigated. Namely, the effect of a non-thermal sub-nanosecond-pulsed electromagnetic field on the aggregation state and functional activity of the enzyme has been studied. The results obtained indicate that a 640 kV/m pulsed electromagnetic field has a certain impact on the HRP enzyme: namely, an increased aggregation of the enzyme is observed after the 40 min incubation of its solution in this field. The change in the aggregation state of HRP, adsorbed onto mica from PEMF-irradiated samples, indicates an alteration in the surface structure of the protein globule, which leads to a change in the interactions between the HRP macromolecules with the mica surface, and with the solvent upon the adsorption. Namely, the following interactions take place in the near-surface layer of the AFM substrate upon adsorption of HRP macromolecules onto mica:  
Figure 5: Results of spectrophotometry-based estimation of HRP enzymatic activity using a standard assay with ABTS. Typical time dependencies of the absorbance at 405 nm obtained for the HRP solution irradiated in PEMF (red curve), and for the control HRP solution (black curve). Experimental conditions: HRP:ABTS:H\({}_{2}\)O\({}_{2}\) = 10\({}^{-9}\) M:0.3 mM:2.5 mM. T = 23 \({}^{\circ}\)C.  
1. HRP monomer--HRP monomer;
2. HRP monomer--mica surface;
3. HRP monomer--solvent (buffer solution). In addition, a change in the protein structure leads to an increase in the equilibrium constants of the above-listed processes. As a result, an increased aggregation of HRP occurs upon its adsorption onto mica. Moreover, the changes in the HRP structure, induced by the action of PEMF, influenced the enzyme's active site conformation, and thus led to a significant (46%) reduction in the HRP functional activity. Moreover, the impact of the pressure jump, induced by a fast rise-time (80 ns) shock wave with a duration of 4 ms, on the HRP enzyme was studied. It has been observed that such a pressure jump can cause a slight change in the HRP aggregation, pronounced in the form of an increase in the number of objects with 2 to 6 nm heights, which have not been observed in the control samples not exposed to the shock wave. That is, the effect of the pulsed pressure on the physicochemical properties of proteins has been shown. It should be noted that in the literature [6; 30; 31; 32; 33], the effect of a much higher (1000 to 3000 bar) constant pressure on the stereochemical properties of heme-containing enzymes was demonstrated. In this way, in [30; 69], such studies with the example of cytochrome P450 were reported; therein, it was shown that, indeed, such an effect takes place, leading to a decrease in the spin state of heme iron in the substrate-bound enzyme--that is, a change in the enzyme's structure near its active site occurs. Analogous studies with the example of HRP were reported in [30; 69], where it was demonstrated that an increased pressure leads to an increase in the rate of HRP association with carbon monoxide. In our present study, we investigated the impact of a pressure jump within a narrow (10 atm) pressure range with a \(\sim\)80 ns rise-time of the front and a short (\(\sim\)4 ms) duration. It was demonstrated that even within this pressure range at such short pulse durations, the pulsed pressure can lead to a change in the physicochemical properties of the enzyme, causing its tendency to aggregate upon adsorption onto mica. External physicochemical impacts can also cause stereochemical changes in the structure of proteins.