MECHANISM OF SALICYLATE HYDROXYLASE REACTION .4. FLUOROMETRIC ANALYSIS OF COMPLEX FORMATION

1. 1.|Binary and ternary complexes between apoenzyme of salicylate hydroxylase (salicylate, NADH: oxygen oxidoreductase (1-hydroxylating, 1-decarboxylating)) and components such as FAD, substrate and NADH were fluorometrically studied to determine dissociation constant and stoichiometric ratio of each component. This was based on the changes in fluorescence intensity when the apoenzyme was mixed with these components. 2. 2.|A protein-denaturing agent or an alkali treatment was shown to depress the fluorescence emission of the apoenzyme with the red shift of the maximum. 3. 3.|The addition of FAD to the apoenzyme resulted in a marked decrease in both FAD and protein fluorescences. 4. 4.|Either substrate or NADH formed 1 :1 complex with the apoenzyme. This was indicated by both quenching of protein fluorescence and enhancement of substrate or NADH fluorescence. 5. 5.|The holoenzyme combined specifically with substrate to form a fluorescent ternary complex in which the ratio of apoenzyme, FAD and substrate was 1:1:1. 6. 6.|Upon formation of these complexes, the blue shift occurred in the fluorescence spectrum of protein, salicylate, NADH or FAD. © 1969.