CONTINUOUS MEASUREMENT OF PHOTOSYNTHETIC 



PHOSPHORYLATION WITH THE FIREFLY 



LUMINESCENCE ASSAY SYSTEM 



B. L. Strehler and D. D. Hendley 



Gerontoloiry Branch, Natiojial Heart Institule, National Institutes of Health, 

 Bethesda, Maryland, and the Baltimore City Hospitals, Baltimore, Maryland 



Introduction 



In 1950, W. A. Arnold and one of the authors (24) attempted 

 to measure ATP formation by illuminated chloroplasts, using the 

 firefly luminescence assay method of Strehler and Totter (25) . This 

 method, which had been successfully used to measure oxidative phos- 

 phorylation in mitochondrial suspensions, is based upon the discovery 

 by McElroy (14) that ATP will support the luminescence of firefly 

 extracts and upon the subsequent description of the properties of 

 the system by McElroy and collaborators (17, 16) . 



Although some stimulation of luminescence was noted when chloro- 

 plasts mixed with firefly extracts were illuminated, it was soon shown 

 that the major effect, at least, was due to a luminescence emitted by 

 the chloroplasts for a few seconds after they were illuminated. This 

 photosynthetic luminescence has been intensively investigated by 

 Arnold, Strehler, Franck, Calvin, and their collaborators (I, 3, 2, 22, 

 7, 10, 27) . 



Subsequent experiments, using appropriate filter combinations, 

 showed an erratic stimulation of firefly luminescence following the 

 illumination of chloroplast-luciferase mixtures, but the reaction 

 could not be studied systematically due to unknown sources of varia- 

 bility. Rather, we concentrated our efforts on the measurement of 

 ATP changes induced in intact Chlorella (21, 23) and on the mechan- 

 ism of photosynthetic luminescence. 



Arnon's unequivocal demonstration of photosynthetic phosphoryla- 

 tion (6) in isolated chloroplasts and the rapid description of the 

 properties and cofactors of the system made feasible a new attempt 

 to measure changes of ATP level during illumination. In the present 

 communication we describe a simple and sensitive method for the 

 instantaneous measurement of ATP levels in illuminated chloro- 



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