¹ Laboratory of Nanobiotechnology, Institute of Biomedical Chemistry, Moscow, Russia

² Laboratory of Shock Wave Impacts, Joint Institute for High Temperatures of Russian Academy of Sciences, Moscow, Russia

³ RES Ltd., Moscow, Russia

⁴ Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk, Belarus

⁵ Foundation of Perspective Technologies and Novations, Moscow, Russia

⁶ Skryabin Moscow State Academy of Veterinary Medicine and Biotechnology, Moscow, Russia

⁷ Department of Infectious Diseases in Children, Faculty of Pediatrics, Pirogov Russian National Research Medical University, Moscow, Russia

The radiothermometry (RTM) study of a cytochrome-containing system (CYP102 A1) has been conducted in order to demonstrate the applicability of RTM for monitoring changes in the functional activity of an enzyme in case of its point mutation. The study has been performed with the example of the wild-type cytochrome (WT) and its mutant type A264K. CYP102 A1 is a nanoscale protein-enzymatic system of about 10 nm in size. RTM uses a radio detector and can record the corresponding brightness temperature (T_br) of the nanoscale enzyme solution within the 3.4–4.2 GHz frequency range during enzyme functioning. It was found that the enzymatic reaction during the lauric acid hydroxylation at the wild-type CYP102 A1 (WT) concentration of ~10⁻⁹ M is accompanied by T_br fluctuations of ~0.5–1 °C. At the same time, no T_br fluctuations are observed for the mutated forms of the enzyme CYP102 A1 (A264K), where one amino acid was replaced. We know that the activity of CYP102 A1 (WT) is ~4 orders of magnitude higher than that of CYP102 A1 (A264K). We therefore concluded that the disappearance of the fluctuation of T_br CYP102 A1 (A264K) is associated with a decrease in the activity of the enzyme. This effect can be used to develop new methods for testing the activity of the enzyme that do not require additional labels and expensive equipment, in comparison with calorimetry and spectral methods. The RTM is beginning to find application in the diagnosis of oncological diseases and for the analysis of biochemical processes.

Keywords: radiothermometry; cytochrome; CYP102 A1; brightness temperature

Radio biology and radio medicine is a field of science that develops the theory and practice of application of radiation for biology and medical purposes [1,2,3,4,5,6,7,8,9,10,11,12,13]. The radiothermometry method is relatively inexpensive and allows one to detect radiation in the microwave range in real-time mode without using additional tags. The RTM method is based on monitoring the T_br, which can change during the biochemical reaction [9]. The T_br is a temperature value that equals the thermodynamical temperature of a complete radiator.

During the biochemical reaction, a non-balanced condition of the medium can appear that is characterized with the increase in T, which can be accompanied by radiation in a certain frequency range. This is why the radio detector used allows us not only to conduct diagnostics to detect tumors on a macrolevel, but also to measure the kinetics of biochemical processes on a milli-level (tumours [3,4,5,6,7,8]), micro-level (cell processes [10]) and even on a nano-level (enzyme reactions, denaturation processes [11,12]) in a microwave range to monitor the changes in T_br.

Regarding the milli-level, the use of RTM for the diagnosis of socially significant diseases was reported in a number of papers: the revelation of cancer at an early stage by detecting an increase in local brightness temperature (in microwave range) in the tumour growth region was reported [1,2,3,4,5,6,7,8]. In addition to standard methods, such as X-ray diagnostics, the RTM method can provide additional “energetic” information on the intensity of proliferative processes and the speed of the tumor growth, etc. It was noted that combining mammography and the RTM method lowers the risk of false negative results three- or four-fold and raises the diagnostics’ sensitivity up to 98% [3].

Regarding micro- and nano-level, studies on the use of radiation occurring during the modulation of functioning of enzymes, either with or without the introduction of microparticle labels, were reported. So, as an example of an approach utilizing labels, the application of micron-sized ferromagnetic iron-oxides for local thermal control of amylase activity with the use of 0.34 MHz radio frequency range can be pointed out [14].

Magnetic nanoparticles of 6 nm to 70 nm in diameter can be employed for the modulation of activity of β-galactosidase, bovine carbonic anhydrase and thermolysin [15,16,17]. An increase in protease activity after the irradiation of the enzyme, labeled with 4 nm gold-coated magnetite particles in a radio frequency (17.76 MHz) field, owing to a conversion of the radio frequency radiation into local heat was demonstrated [18]. Moreover, a very interesting approach to the local regulation of the properties of proteins with the use of iron oxide nanoparticles of various sizes was recently reported by Ovejero et al. [19]. These authors developed a selective magnetic nanoheating approach for the multi-hot-spot induction and sequential regulation of enzymes [19]. This approach creates a new paradigm, providing an opportunity for the selective regulation of multi-enzyme reactions.

Within the range of shorter microwaves, modulation of the properties of proteins was demonstrated even without the introduction of nanometer-size particles. So, electromagnetic irradiation was demonstrated to induce conformational transitions in hemoglobin [20]. The possibility of conformational changes in macromolecules upon the impact of electromagnetic radiation was shown with the example of antibody/antigen interaction [21].

On a nanoscale, studies on the registration of self-radiation of label-free enzyme systems are important. It is known that nanoscale heme-containing enzymatic systems based on horseradish peroxidase (HRP) and CYP102 A1, where enzymatic components have the size of about 10 nm, as noted above [22], can emit in the microwave band as they function [9,12,23]. In these studies, radiation was detected using the RTM method in the microwave range of 3.4 to 4.2 GHz. The question is, however, whether or not there can be a change in the microwave radiation of enzyme systems upon point mutations in the enzymes. This is particularly important, since the increased expression of the mutant forms of proteins is known to take place in some cases of oncological diseases [13,24,25]. We should note that cytochromes P450 and their mutant forms can take part in cancer formation and cancer treatment. They mediate metabolic activation of numerous pre-carcinogens and participate in the activation and inactivation of anti-tumor medicines [26]. This makes developing and applying new methods to analyze the functional condition of these systems in order to receive fuller, more relevant information about them.

To study the mechanism of cytochrome P450 functioning, a simpler model bacterial system CYP102A1 of the cytochrome P450 enzyme superfamily characterized in [22] is usually employed, and, in the present study, we have also used this model system. CYP102A1 represents a self-sufficient enzyme, where reductase and heme domains are linked in one polypeptide chain [23]. The latter fact raises interest in it as a convenient simplified model of a transport chain of monooxygenase systems containing cytochrome P450, which plays a role in CYP102A1 catalysis of hydroxylation of saturated and unsaturated fatty acids [27]. As we demonstrated previously, during the functioning of an enzyme system containing 10⁻⁹ M of the CYP102 A1 enzyme, microwave radiation has been observed in the form of T_br pulses [9].

In the present work, the registration of the influence of the effect of a point mutation in cytochrome P450 CYP102A1 on the brightness temperature in a model reconstructed system. For this purpose, we compared Tbr fluctuation for the wild type of CYP102 A1 (WT) and its mutant type CYP102 A1 (A264K). The activity of CYP102 A1 (WT) in the presence of the lauric acid (LA) is k_cat = 50 s⁻¹ at the enzyme concentration of ~10⁻⁹ M [28]. In addition to this, we know that the activity of the mutant type CYP102 A1 (A264K) upon lauric acid hydroxylation is about k_cat = 0.006 s⁻¹ [29], which is three orders lower than the activity exhibited by CYP102 A1 (WT). Therefore, we have chosen CYP102 A1 (A264K) for our study in order to conduct a comparative analysis of radiation of solutions in the presence of a significantly less active CYP102 A1, but at the same enzyme concentration. We have shown that the passage from the wild type of protein to its mutant type connected with an 8333-fold increase in activity leads to the disappearance of microwave radiation. Thus, we have shown that the Tbr fluctuation of the solution CYP102 A1 is dependent on its activity

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Ivanov, Y.D.; Malsagova, K.A.; Bukharina, N.S.; Vesnin, S.G.; Usanov, S.A.; Tatur, V.Y.; Lukyanitsa, A.A.; Ivanova, N.D.; Konev, V.A.; Ziborov, V.S. Radiothermometric Study of the Effect of Amino Acid Mutation on the Characteristics of the Enzymatic System. Diagnostics 2022, 12, 943.

Diagnostics 2022, 12(4), 943; https://doi.org/10.3390/diagnostics12040943