We propose to examine the interaction of ATP and its metal chelates with enzymes which utilize ATP as a substrate. The enzyme of particular interest to us in nitrogenase for which ATP hydrolysis is an obligatory part of the overall enzyme reaction. ATP binds to component II of nitrogenase in the presence of Mg to induce an EPR detectable conformation change and to alter the redox potential of the component. Electrons are transferred from component II to I to reducible substrate, with the hydrolysis of ATP. We will modify the method of direct scanning of gel chromatography columns by using a rapid transport for the optical system and a rapid data acquisition system, so that we can observe changes of hydrodynamic properties of proteins which occur on a time scale of seconds to minutes. Interaction of Mg ATP and Cr ATP with hexokinase will be used as a tractable model system for development of the gel scanning system. A conformation change is induced in hexokinase by Cr ATP and lyxose, which are very poor substrates; this change may be detectable by direct gel scanning methods. Direct gel scanning will then be used to examine the interaction of ATP with component II of nitrogenase in the presence and absence of Mg. Similar experiments will be carried out using other variables including pH, ionic strength, and reductant concentration, all of which are known to alter the EPR visible conformation change of component II. Direct gel scanning will also be used to examine the interactions of components I and II in saturation, and large zone and small zone chromatography experiments. Methods will be developed for chromatography of functioning enzyme, and direct scanning will be applied to such chromatography.