FLUORESCENCE STUDIES OF COENZYME BINDING TO DEHYDROGENASES

Publisher Website
Google Scholar
Abstract
The fluores-cence spectrum of reduced DPN is not altered when the compound is added to crystalline beef heart lactic dehydrogenase but a very marked increase in the intensity of the fluorescence of the reduced acetyl pyridine analog of DPN is observed when this compound is added to the crystalline enzymes. The increase in fluorescence intensity is associated with a shift in the fluorescence maximum of the analog and is dependent on the amount of lactic dehydrogenase added to the system. The differences observed between the activity of reduced DPN and the reduced acetyl pyridine analog are in accordance with the spectro-photometric observations. Using the fluorescence procedure, the bind-ing of lactic dehydrogenase with the reduced coenzyme analog was determined with only a few micrograms of protein. When activated at 288 m[mu], crystalline beef heart lactic dehydrogenase gives a fluorescence maximum at 350 m[mu], and this fluorescence pattern can not be reproduced by a mixture of amino acids. Treatment by agents which produce reversible and irreversible denaturation of the enzyme or addition of the reduced acetyl pyridine analog of DPN to the system causes a loss of the fluorescent characteristics of the lactic dehydrogenase. The significance of the fluorescence changes associated with coenzyme binding is briefly discussed.