Reduction of a disulphide in relation to the metabolic states of mitochondria

The role of -SH groups in mitochondrial energy transfer reaction was studied by observing the reduction of a disulphide, 5, 5'-dithiobis (2-nitrobenzoic acid), DTNB, a specific analytical agent for the estimation of -SH groups in biological materials, by addition of it to the isolated rat liver mitochondria in various respiratory states, as defined by CHANCE and WILLIAMS. 1. In the various respiratory states, states 1 to 5, the reduction of DTNB proceeds most rapidly at state 5, and most slowly at state 3. DTNB reduction at state 5 is suppressed by the partial oxidation of respiratory carriers with oxygen (state 4) and the addition of respiratory substrate does not affect the DTNB reduction. 2. The retardation in the reduction rate at state 3 is relieved partially by a respiratory inhibitor, KCN, and is intensified markedly by oligomycin, an inhibitor of oxidative phosphorylation. An uncoupler for oxidative phosphorylation, DNP, does not affect the reduction rate at state 3. At state 4 the reduction is stimulated by DNP and KCN, but is unaffected by oligomycin. The results suggest that the alteration in the functions of the energy transfer reaction in mitochondria is accompanied by changes in the occurrence and the functioning of -SH groups which can be detected by the reactivity with DTNB. The data suggest also that there are at least two kinds of -SH groups reacting with DTNB: the one is the -SH group which reacts DTNB actively when the respiratory carriers are kept reduced, and the other is the one which reacts actively when the respiratory carriers are kept oxidized, participating in the phosphorylating system and its reactivity with DTNB diminishes in the actively phosphorylating states (states 2 and 3).