Understanding tetrazolium reduction and the importance of substrates in measuring respiratory electron transport activity

Most of the oxygen consumption in plankton is controlled by an enzymatic complex called the electron transport system (ETS) or the electron transport chain. To detect and measure this ETS in a biologically diverse plankton community it is common practice to add, in addition to an artificial electron acceptor, the various substrates that donate reducing equivalents to the ETS. Specifically, pyridine nucleotides (nicotine adenine dinucleotide (NADH) and nicotine adenine dinucleotide phosphate (NADPH)) and succinate are routinely used. The addition of these substrates to the ETS stimulates its activity to capacity and hence serves as a measure of potential respiration (Φ). This proxy is then used in ecological and oceanographic studies as an index for respiration. Unfortunately in an attempt to align this proxy more closely with in vivo physiological respiration and to simplify the ETS assay the required substrates were omitted from the analysis. The consequences of this shortcut are demonstrated and explained here. In effect, because some basic rules of biochemistry were broken, the simplified assay yields misleading results that are, in fact, the equivalent of the control (the blank) in a normal ETS assay. Here we explain some of the chemistry of this background reaction, most of which can be found in textbooks. We demonstrate that the use of the artificial electron acceptor, the tetrazolium salt, INT, is not specific for cell respiratory ETS reactions, but is sensitive to many, substances present in all cells. We show that, among these common cellular substances, ascorbic acid, cysteine, and glutathione readily reduce INT non enzymatically to its formazan. Such INT reduction is part of the normal blank reaction in the ETS assay and is far removed from the desired respiratory capacity of the cell.

► Assaying ETS activity without substrates detects background reduced organics. ► Ascorbic acid, cysteine, glutathione strongly reduce the tetrazolium salt, INT. ► The reduction of tetrazolium salts is not specific for respiratory enzymes. ► Without substrate addition, formazan production is reduced more than 40 times.