The effect of oxygen tension on tetrazolium reduction by respiratory enzyme systems of tissue culture cells

1. Attempts have been made to confirm how the formazan formation is affected in the presence of oxygen gas when the cells are incubated with neotetrazolium salt and the subsrtates for the enzymes to be tested. 2. In the cases of succinoxidase formazan formation is minimized under pure O2tension, it increases with decrease in O2 tension, and reaches its maximum value under N2gas. 3. This relationship between the oxygen tension and the diformazan formmation can likewise be observed even after pretreatment of the system with KCN. 4. In measuring enzyme activity of the DPN-diaphorase system with L-glutamate and DPN as substrate and NT as hydrogen acceptor, the same relationships between the oxygen tension and the NT-reduction can be seen as in succinoxidase system. 5. In the determination of enzyme activity of the cytochrome-c-cytochrome oxidase system with p-phenylene-diamine as substrate and NT as hydrogen acceptor, likewise the diformazan formation is markedly affected by oxygen tension and increased with the reduced oxygen tension but under pure Ns gas the value is reduced. When the systen is pretreated with KCN, however, the diformazan formation reveals its maximum value under pure nitrogen gas, the values of which correspond to those values of endogenous reaction without substrate. 6. The above results show that the neotetrazolium salt can compete with O2 as hydrogen acceptor, and less values of formazan formation may be obtained under higher oxygen tension and the higher values under lower oxygen tension independently from the true activity of the enzyme.