## 4 Discussion  
This study looks into how glycerol flow through a polymeric pipe affects the properties of a model enzyme protein, the solution of which was placed near either the inflow or the outflow sections of the pipe. We have shown that after placing the enzyme solution near the linear sections of the pipe with flowing glycerol, changes in its properties are observed. Namely, after the HRP solution was incubated near the pipe inflow, a decreased adsorption of the protein onto mica was observed, being approximately 5.5 times lower than that observed for the control solution incubated far away from the pipe. At that, the enzymatic activity of the protein did not change. On the contrary, in the case when the HRP solution was incubated near the pipe outflow, an increased (approximately 1.6 times higher) protein adsorption was observed, while the enzymatic activity decreased considerably (by approximately 18%). The data obtained indicate that the glycerol flow affects the physicochemical properties of HRP macromolecules. At that, the effect of the flow in different sections of the pipe varies. This is supposed to be caused by a difference in the intensity of the influence, which leads to variations in the character of protein structure deformations. Thus, a decreased protein adsorption in the case when the solution was incubated near the inflow pipe section  
Figure 4: Spectrophotometry-based estimation of HRP enzymatic activity. Characteristic time dependencies of change in solution absorbance at 405 nm obtained for control HRP sample and for HRP samples incubated in the experimental setup. The measuring cell (containing HRP solution) was placed either far away at a 10-m distance from the experimental setup (control experiment, black line), near the inflow (blue line) or near the outflow (red line) section of the pipe. Experimental conditions: HRP:ABTS:H\({}_{2}\)O\({}_{2}\) = 10\({}^{-9}\) M:3 mM:2.5 mM. T = 23 \({}^{\circ}\)C.  
is apparently caused by a decrease in electrostatic interaction between negatively charged surface groups of the mica substrate (whose zeta potential makes up \(-\)75 mV [31]) and positively charged groups on the surface of HRP (which represents a basic protein with an isoelectric point of about 9 [32]). Electrostatic interactions often play a governing role in protein adsorption [33]. Accordingly, the weaker the electrostatic interaction between the protein molecules and the substrate surface is, the lower the amount of the surface-adsorbed protein. Another cause of the observed decrease in the HRP adsorption can consist in an increased contribution of repulsion forces between negatively charged surface groups of the substrate and negatively charged groups of the protein globule. That is, an effect of a change in the local structure of the protein globule's surface areas, which determine its interaction with the substrate surface, has been revealed. At that, the spectrophotometry data indicate no change in the protein's functional activity. This indicates that the changes in the enzyme protein structure do not affect its active site. In another case, when the HRP solution was incubated near the outflow section of the pipe, an increase in the number of objects on the substrate surface was observed, whereas their height increased from 1 nm up to 2 to 2.5 nm. That is, both protein adsorption properties and its aggregation state have changed (the content of aggregates has increased). Moreover, the spectrophotometry measurements also revealed a change in the HRP functional activity, which was found to decrease in comparison with the control experiment. This is why we can assume a different influence of glycerol flow on the protein in this case. This influence leads to a different kind of protein structure change, which affects not only the surface of the enzyme globule, but also its active site. The data on the effect of the glycerol flow through the pipe on the properties of a protein are confirmed by the results obtained in our previous studies.