Biomedical Chemistry: Research and Methods 2025, 8(2), e00276

SUPEROXIDIZING AND ANTIOXIDANT ACTIVITY OF NICOTINAMIDE COENZYMES IN VITRO

T.V. Sirota*, M.V. Akulenko, N.P. Sirota

Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Institutskaya ul. 3, Pushchino, Moscow Region, 142290, Russia; *e-mail: sirotatv@rambler.ru

Keywords: nicotinamide coenzyme; NAD; NADP; NADH; NADPH; superoxide, nitroblue tetrazolium

DOI:10.18097/BMCRM00276

The whole version of this paper is available in Russian.

New properties of nicotinamide coenzymes (NAD, NADP, NADH, NADPH) have been discovered: superoxide-generating and antioxidant activities. Coenzymes are capable of generating superoxide anions (O2─●), entering an alkaline environment; and can be O2─● traps, inhibiting the generation process in superoxide-generating model systems, thus exhibiting antioxidant properties. Actually, nicotinamide itself, which is a functional part in the coenzyme molecule in oxidation-reduction processes, showed only antioxidant activity in vitro. Antioxidant properties have also been found in adenosine, which is part of the coenzyme molecule. Thus, it has been established for the first time that coenzymes, according to their chemical properties, are bifunctional molecules. It is assumed that they participate in cellular signaling through the generation of superoxide and, in the case of excess superoxide in the environment, they can be antioxidants. All these properties of coenzymes and their components must be taken into account when used in scientific research and medicine.
Figure 1. Structural formulas of the oxidized and reduced forms of coenzymes.

Figure 2. Kinetics of diformazan formation in 0.2 M carbonate buffer, pH 10.8, containing 0.075 mM NBT in the presence of: 1 - 1.5 mM NAD and 2 – continued recording; 3 - 1.5 mM NADH; 4 - 1.5 mM NADP. Temperature 22°C.

Figure 3. Effect of SOD on superoxide generation with the participation of 1 mM NADH: 1 – enzyme, 0.92 μg protein/ml, present in 0.2 M carbonate buffer, pH 11.35, containing 0.075 mM NBT; 2 – control: buffer and NADH.

Figure 4. Effect of different NADPH concentrations on the kinetics of diformazan formation in the superoxide-generating reaction xanthine-xanthine oxidase: 1 – 1.0 mM; 2 – 0.5 mM: 3 – control. Measurement conditions: 50 mM carbonate buffer, pH 10.2, containing 0.4 mM EDTA, 0.1 mM xanthine, 0.025 mM NBT, xanthine oxidase 11 μg protein/ml. Temperature 19°C.

Figure 5. Comparison of the antioxidant effect of NADH and NAD on the kinetics of diformazan formation in the reaction of adrenaline autoxidation: 1 – 1.0 mM NADH; 2 – 1.0 mM NAD; 3 – control. Reaction conditions: 0.2 M carbonate buffer, pH 10.6; 0.05 mM NBT, 0.058 mM adrenaline. Temperature 20°C.

Figure 6. Antioxidant activity of different NADH concentrations. Measurement conditions: 0.2 M carbonate buffer, pH 10.6; 0.05 mM NBT, 0.058 mM adrenaline. Temperature 20°C.

CLOSE
Table 1. Antioxidant activity of the studied compounds in the enzymatic reaction xanthine-xanthine oxidase.

FUNDING

The work was carried out within the framework of the State Assignment of ITEB RAS No. 075-00223-25-00.

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