1 Institute of Biomedical Chemistry (IBMC), Moscow, Russia
2 Joint Institute for High Temperatures of Russian Academy of Sciences, Moscow, Russia
3 RES Ltd, Moscow, Russia
4 Foundation of Perspective Technologies and Novations, Moscow, Russia
5 Skryabin Moscow State Academy of Veterinary Medicine and Biotechnology, Moscow, Russia
The concentration dependence of a microwave frequency radiation from a solution of a functioning enzyme system (ES) (with the example of cytochrome P450 BM3 (CYP102A1) system during lauric acid (LA) hydroxylation) has been studied with a radiothermometric sensor. Registration of the radiation from the enzyme solution has been performed in the frequency range from 3.4 to 4.2 GHz at the enzyme concentrations from 10−10М to 10−6М. It has been demonstrated that the catalysis of LA hydroxylation in a reconstituted CYP102A1 system is accompanied by a generation of microwave radiation over the entire range of concentrations studied. It has been found that a transition from a multipulse mode (at nanomolar enzyme concentrations from 10−10М to 10−8М) to a single-pulse mode (at micromolar enzyme concentrations from 10−7М to 10−6М) is observed. This effect is discussed on the basis of assumptions considering possible realization of biomaser-like radiation in the enzyme system. The discovered concentration-based effect of the transition of an unsynchronized pulsed radiation into a synchronized one in ES can further be used in the development of novel methods of noninvasive diagnostics of diseases, in mathematical modeling of the functioning of living systems, and in the development of next-generation quantum computers.
Functioning of enzyme systems (ES) can be considered as a cyclic transition of the system from a nonexcited state to an excited nonequilibrium one and then back to the equilibrium. During the process of catalysis, a visible-range radiation can be observed for a number of enzyme systems, e.g., for the oxidative enzymes that catalyze the reaction followed by bioluminescence, i.e., luciferase . Radiation from enzyme systems can be observed in other spectral ranges as well. In this way, previously it was demonstrated that the functioning of the CYP102A1 enzyme system in a narrow concentration range (from 10−9М to 10−8М) is accompanied by a microwave radiation in the form of pulse trains at 3.4 to 4.2 GHz frequency . In , a radiothermometry method was employed. This method is becoming more common for monitoring of functional state of both cellular systems and the whole organism [3, 4]. Radiothermometer (abbreviated, radiometer) measures the object’s brightness temperature Tbr. The brightness temperature (radio temperature) is the temperature value that is numerically equal to the thermodynamic temperature of an ideal black body. In the process of catalytic reaction, a nonequilibrium state of the medium can occur; this state is characterized by an increase in the brightness temperature, what can be accompanied by a radiation in a certain frequency range connected with the increase in the brightness temperature. Thus, the use of a radiometer operating in a microwave range allows one to measure the kinetics of biochemical processes by monitoring changes in the brightness temperature. This approach is very convenient, as it is label-free, does not require expensive equipment, and provides new information about quantum phenomena in biological systems. Such processes of increase in brightness temperature accompanied by radiation in various spectral ranges, often manifest themselves not only in natural biological systems, but are also observed in gas-discharge devices (including domestic ones), during nonequilibrium processes in shock waves, upon flowing of liquid media through injectors, etc.
The CYP102A1 system was selected to be used in the study in  owing to the fact that bacterial enzyme CYP102A1, which pertains to the super family of heme-containing cytochromes P450, plays a crucial role in Bacillus megaterium enzymatic pathways . This protein represents a self-sufficient enzyme requiring no any extra partner proteins for the implementation of a catalytic cycle. The enzyme is structured so that its reductase and heme domains are combined in a single polypeptide chain . This stimulates the interest in studying this enzyme as a convenient simplified model of cytochrome P450-containing monooxygenase systems, contributing to oxidation of endogenous and exogenous substrates in an organism . CYP102A1 catalyzes hydroxylation (mainly, (ω-1), (ω-2), and (ω-3)–hydroxylation) of saturated and unsaturated fatty acids with various chain lengths . It was also demonstrated that full-length CYP102A1 can exist in both monomeric and oligomeric states, primarily in the form of dimers (>50%) . Besides, dimeric form exhibits higher activity in comparison with the monomeric one, with kcat of 50s−1 and 10s−1 for the dimers and monomers, respectively .
The objective of our study is to measure the dependence of a radiation from the CYP102A1 enzyme system on the enzyme concentration (С) in a wide range of concentrations from С=10−6М to С=10−11М in the process of lauric acid (LA) substrate hydroxylation. A wider concentration range (as compared to the one studied earlier in ) has been studied for the following reasons.
(1) The concentration range below 10−9М corresponds to the conditions of reduced enzymatic activity of CYP102A1. A shift of the equilibrium of the enzyme dimerization towards the formation of monomers with lower activity can be observed within this range .
(2) The enzyme concentration greater than 10−8М corresponds to the concentration level of proteins in a cell.
Within the framework of the present study, a dependence of microwave radiation from the CYP102A1 enzyme system on the enzyme concentration in the solution has been studied in the process of catalytic cycle of the enzyme by monitoring the brightness temperature with a microwave radiometer sensor.
It has been demonstrated that the functioning of CYP102A1 enzyme system is accompanied by a microwave radiation within the frequency range from 3.4 to 4.2 GHz at enzyme concentrations from С=10−10М to C=10−6М. Moreover, the radiation has appeared to be concentration-dependent. In this way, at high enzyme concentration (~10−6М), the radiation from the ES had the form of single sync pulses correlated to the time of stirring, i.e., mechanical excitation of the system. At lower enzyme concentration, the radiation had the form of pulse trains. At the enzyme concentration of 10−11М, no radiation has been registered.
Journal of Sensors, Volume 2019, Article ID 7608512, 11 pages