Diethylbarbiturate potentiation of 2,4,6-trinitrobenzenesulphonate-induced rhodanese inactivation

The rate of rhodanese inactivation by 2,4,6-trinitrobenzenesulphonate is increased in the presence of diethylbarbiturate in the reaction medium. A "rate saturation effect" indicates the formation of a rhodanese-diethylbarbiturate complex, prior to modification-induced enzyme inactivation. The dissociation constant of this complex is 19.0 mM. Diethylbarbiturate has no effect on the trinitrophenylation rate of the free amino groups of rhodanese. When rhodanese modification, in the presence of diethylbarbiturate in the reaction medium, is carried out by the use of a 2,4,6-trinitrobenzenesulphonate concentration much lower than the concentration of rhodanese modifiable amino groups, reaction stoichiometry indicates that 3 to 5 moles of rhodanese are rendered inactive for each mole of 2,4,6-trinitrobenzenesulphonate utilized. This finding indicates the existence of a chain-reaction type mechanism of rhodanese inactivation.