Biomedical Chemistry: Research and Methods 2023, 6(1), e00188

Superoxide Generation by Nicotinamide Coenzymes

T.V. Sirota

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

Keywords: superoxide; nitro blue tetrazolium; diformazan; nicotinamide coenzymes; NADPH; NADP; NADH; NAD

DOI:10.18097/BMCRM00188

The whole version of this paper is available in Russian.

Alkaline solutions of nicotinamide coenzymes can generate superoxide radicals.  Their formation was registered by reduction of nitroblue tetrazolium (NBT) present in the buffer with formation of diformasan. Inhibition of diformazan formation occurs when superoxide dimutase (SOD) is added to the system, thus confirming generation of О2─●. The highest superoxide generating activity was observed with NADPH. In the case of NADPH and NADH, the rate of superoxide generation was significantly lower (by approximately 50%). No О2─● was detected when NAD was used under the same conditions and in the same time; however, 4 h later, diformasan was detected in the same sample. The superoxide generating activity decreased in the following order: NADPH > NADH ≥ NADP > NAD. Other compounds tested (adenosine, ADP and ATP) did not generate superoxide radicals even after prolonged incubation. In a cell, where a local changes in the pH of the environment are possible, nicotinamide coenzymes can be potential sources of О2─● and thus participate in cell signaling. A change in pH can initiate this process.
Figure 1. A - Kinetics of diformazan formation in 0.2 M carbonate buffer, pH 10.8, containing 0.075 mM HBT in the presence of 1.5 mM concentration of the corresponding coenzyme: 1 - NADPH,
2 - NADH. Controls: no NBT in the buffer and the following components are added: 3 - NADPH,
4 - NADP; 5 - only HBT in the buffer and no nicotinamide coenzymes.
B - The effect of SOD on the generation of superoxide with the participation of NADH: 1- NADH is added to the buffer; 2 - the same in the presence of SOD, 0.92 µg protein/ml. Reaction conditions as in Fig. 1 A.
Figure 2. Spectra of 0.1 mM NADPH (curve 1) and 0.1 mM NADP (curve 2) in bidistilled water.
Figure 3. Spectra of reaction products after a 10-minute presence of NADP or NADPH in 0.2 M carbonate buffer, pH 11.3, containing 0.075 mM HBT: 1 - products of 1.5 mM NADP; 2 - products of 1.5 mM NADPH; 3 – after 1:1 dilution with bidistilled water of NADPH products.

CLOSE
Table 1. Superoxide generating activity of the studied compounds.

FUNDING

The work was carried out within the framework of the State assignment ITEB RAS No. 075-01027-22-00

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