Detection of microwave radiation of cytochrome CYP102 A1 solution during the enzyme reaction

Abstract

Microwave radiation at 3.4-4.2 GHz frequency of the cytochrome P450 CYP102 A1 (BM3) solution was registered during the lauric acid hydroxylation reaction. The microwave radiation generation was shown to occur following the addition of electron donor NADPH to a system containing an enzyme and a substrate. The radiation occurs for the enzyme solutions with enzyme concentrations of 10^-8 and 10^-9 М. The microwave radiation effect elicited by the aqueous enzyme solution was observed for the first time. The results obtained can be used to elaborate a new approach to enzyme systems research, including studying of the mechanism of interaction of a functioning enzyme system with microenvironment.

Keywords: Cytochrome P450; Microwave radiation; Monooxygenase system.

Fig. 1

Results of control experiments: brightness temperature as a function of time Т_b(t). Experimental conditions of the control experiments:

1. –

   Temperature of measurements 18 °С (dotted lines) and 23 °С (solid lines);

2. –

   (blue lines) the solution under analysis contains protein CYP102 A1 (10⁻⁹ M) and substrate (lauric acid, 0.5 mM) (variant 1);

3. –

   (red lines) the solution under analysis contains substrate (lauric acid 0.5 mM) upon addition of electron donor (NADPH, 0.2 mM) (variant 2).

4. –

   (black line) the solution under analysis contains protein CYP102 A1 (10⁻⁹ M) (variant 3);

Arrows indicate the time points of addition of electron donor NADPH (variants 1 and 2) and H₂O (variant 3) to the system containing protein and substrate, and time points of stirring the solution in the cell.

Fig. 2

Results represent the function of Т_b(t) for cytochrome CYP102 A1 solution at 23 °С. Experimental conditions:

1. –

   cytochrome CYP102 A1 concentration 10⁻⁸ М (blue line) and 10⁻⁹ М (red line);

2. –

   the temperature of solution under analysis is 23 °С;

Arrows indicate the time points of the addition of electron donor (NADPH, 0.2 mM) to the system containing protein and substrate (lauric acid, 0.5 mM), and the time points of stirring the solution in the cell.

Fig. 3

Results represent the function of Т_b(t) for cytochrome CYP102 A1 solution at 18 °С. Experimental conditions:

1. –

   cytochrome CYP102 A1 concentration 10⁻⁸ М (blue line) and 10⁻⁹ М (red line);

2. –

   the temperature of solution under analysis is 18 °С;

Arrows indicate time points of addition of electron donor NADPH to the system containing protein and substrate, as well as time points of stirring the solution in the cell.

Fig. 4

The results of the comparative experiments. Experimental conditions:

1. –

   the temperature of solution under analysis is 23 °С;

2. –

   (blue line) the solution under analysis contains protein CYP102 A1 (wild type, 10⁻⁹ M) and substrate (hexane, 1% v/v);

3. –

   (red line) the solution under analysis contains protein CYP102 A1 (mutant form, 10⁻⁹ M) and substrate (lauric acid, 0.5 mM);

Arrows indicate time points of addition of electron donor NADPH to the system containing protein and substrate, as well as time points of stirring the solution in the cell.