AFM study of changes in properties of horseradish peroxidase

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 pyramid’s apex or in the center of its base influences the physicochemical properties of the protein macromolecules.

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Fig: Typical AFM images (a–c) and cross-section profiles (d–f) of mica-adsorbed HRP, obtained in control experiments (a,d) and after 40-min incubation of the HRP solution in the vicinity of the apex of the pyramid (b,e) or in the center of its base (c,f). The cross-section profiles correspond to the lines shown in the respective AFM images. The images were obtained using NOVA Px software (ver. Nova_3.4_20014; NT-MDT, Moscow, Zelenograd, Russia).

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.

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

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