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Yuri D. Ivanov, Tatyana O. Pleshakova, Ivan D. Shumov, Andrey F. Kozlov, Irina A. Ivanova, Anastasia A. Valueva, Maria O. Ershova, Vadim Yu. Tatur, Igor N. Stepanov, Victor V. Repnikov, Vadim S. Ziborov
AFM study of changes in properties of horseradish peroxidase after incubation of its solution near a pyramidal structure

O - Yuri D. Ivanov 1,2
O - Tatyana O. Pleshakova 1
O - Ivan D. Shumov 1
O - Andrey F. Kozlov 1
O - Irina A. Ivanova 1
O - Anastasia A. Valueva 1
O - Maria O. Ershova 1
O - Vadim S. Ziborov 1,2
O - Vadim Yu. Tatur 3
O - Igor N. Stepanov 3
O - Victor V. Repnikov 4


1. Institute of Biomedical Chemistry, Moscow, Russia

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

3. Foundation of Perspective Technologies and Novations, Moscow, Russia

4. Bruker Ltd., Moscow, Russia


Abstract

In our present paper, the influence of a pyramidal structure on physicochemical properties of a protein in buffer solution has been studied. The pyramidal structure employed herein was similar to those produced industrially for anechoic chambers. Pyramidal structures are also used as elements of biosensors. Herein, horseradish peroxidase (HRP) enzyme was used as a model protein. HRP macromolecules were adsorbed from their solution onto an atomically smooth mica substrate, and then visualized by atomic force microscopy (AFM). In parallel, the enzymatic activity of HRP was estimated by conventional spectrophotometry. Additionally, attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) has been employed in order to find out whether or not the protein secondary structure changes after the incubation of its solution either near the apex of a pyramid or in the center of its base. Using AFM, we have demonstrated that the incubation of the protein solution either in the vicinity of the pyramids apex or in the center of its base influences the physicochemical properties of the protein macromolecules. Namely, the incubation of the HRP solution in the vicinity of the top of the pyramidal structure has been shown to lead to an increase in the efficiency of the HRP adsorption onto mica. Moreover, after the incubation of the HRP solution either near the top of the pyramid or in the center of its base, the HRP macromolecules adsorb onto the mica surface predominantly in monomeric form. At that, the enzymatic activity of HRP does not change. The results of our present study are useful to be taken into account in the development of novel biosensor devices (including those for the diagnosis of cancer in humans), in which pyramidal structures are employed as sensor, noise suppression or construction elements.


Introduction

Studying pyramidal structures attracts growing interest owing to a concentration of electromagnetic radiation near the apex and base of these structuresas was demonstrated by Balezin et al.1. Moreover, the use of pyramidal structures in biosensor devices was demonstrated2. Pyramidal structures are also employed in atomic force microscopy (AFM)-based highly sensitive sensors with probes of pyramidal shape3. Shielded equipment cabinets or even whole rooms, based on anechoic chambers, are widely employed in order to decrease external electromagnetic impacts on both the electronic sensors and the biological systems under study. Such chambers are fabricated on the basis of acanthoid structures, including pyramidal ones4,5,6. As a rule, these structures are organized in the form of an ordered set of pyramids, or in the form of a multilayer system of pyramids5. Some computational studies, considering the electric noise reduction effects of various structures, were reported7; therein, it was shown that a spatial change in the structure of an electric field occurs owing to both the reflection from the pyramids' surface and the damping in pyramids' material. In biological experimental studies, pyramidal elements can also be employedfor instance, as supporting or directional elements for positioning measuring cells in the sensor devices, or as sensor elements. The use of pyramidal elements can influence the spatial distribution of an electromagnetic field and, accordingly, have an effect on both the biological objects under study and the electronic measuring equipment. Thus, the influence of external electromagnetic radiation, concentrated or scattered by such pyramidal elements, on biological objects in analytical systems represents an important problem of modern biomedical research.

Herein, the influence of a single pyramidal structure on the physicochemical properties of a protein in solution has been investigated. In our experiments, a structure similar to the industrially produced pyramidal structures8,9, has been employed. Samples of a buffered solution of horseradish peroxidase protein were placed and incubated in various positions relative to the pyramidal structure. After the incubation, the HRP macromolecules were adsorbed from the sample solutions onto an atomically smooth surface of bare mica substrates, and visualized on this surface by AFM. The heights of the AFM images of the visualized macromolecules and their amount on the substrate surface were estimated. The use of HRP enzyme in the study was motivated by the fact that this protein is comprehensively characterized in the literature, thus being widely employed as a model in biomedical research10.

As was demonstrated in our previous study, atomic force microscopy (AFM) allows one to reveal the effect of electromagnetic fields on physicochemical properties of proteinsin particular, on their aggregation state and adsorbability onto solid substrate surfaces10. It should be emphasized that in the latter paper10, the electromagnetic field was generated intentionally. At the same time, electromagnetic fields of various intensities and frequencies are now extensively used in both laboratory practice and everyday life. These fields can concentrate at the expense of a resonance effect from various resonator structures, as was demonstrated by Balezin et al.1. These structures form specific spatial topology of electromagnetic field1. The effect of these external electromagnetic fields on living systemsincluding enzymescan depend on the surrounding environment.

Herein, AFM has been employed to find out whether the incubation of the solution of horseradish peroxidase enzyme protein in certain locations in relation to a pyramidal structure (namely, in the vicinity of the apex of the pyramid and in the center of its base) under conventional biochemical laboratory conditions affects the proteins properties. Alterations in the structural properties of biological macromolecules can induce changes in their functionality (one of the examples is myeloperoxidase, which is only functional in dimeric form11). Generally, aggregation of a protein often leads to loss in its functionality12. Accordingly, the results obtained are useful to be taken into account in the development of highly sensitive biosensors, in which proteins are analyzed or employed as surface-immobilized ligands. In highly sensitive biosensor systems, the measurements are performed at the level of single molecules, providing ultra-high detection sensitivity. Application of such systems will allow one to solve a number of important problems of biomedicine, including early diagnosis of diseases in humans. In this respect, however, biomedical applications of single-molecule measurements are reported in pioneer studies. Thus, all factors, including external electromagnetic fields, which potentially may influence the measurement results, must be thoroughly studied.


Full text in format PDF (2563)

Ivanov, Y.D., Pleshakova, T.O., Shumov, I.D. et al. AFM study of changes in properties of horseradish peroxidase after incubation of its solution near a pyramidal structure. Sci Rep 11, 9907 (2021). https://doi.org/10.1038/s41598-021-89377-z


Scientific Reports volume 11, Article number: 9907 (2021)

Nature



Yuri D. Ivanov, Tatyana O. Pleshakova, Ivan D. Shumov, Andrey F. Kozlov, Irina A. Ivanova, Anastasia A. Valueva, Maria O. Ershova, Vadim Yu. Tatur, Igor N. Stepanov, Victor V. Repnikov, Vadim S. Ziborov, AFM study of changes in properties of horseradish peroxidase after incubation of its solution near a pyramidal structure // « », ., 77-6567, .27136, 11.05.2021

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