Luminescence of chlorophyll in spinach chloroplasts induced by acid-base transition.

The occurrence of a delayed light emission in photosynthetic organisms was first reported by Strehler and Arnold.' This emission was shown to have the same spectrum as that of the fluorescence of chlorophyll in vivo,2 but a much greater lifetime. It therefore reflects the restoration of singlet excited states in chlorophyll at the expense of metastable energy. Arnold and Sherwood3 have proposed a semiconductor mechanism in the chloroplast to explain the delayed light emission, whereas Strehler4 has favored a mechanism based on back-reaction of chemical intermediates of photosynthesis. Observations in this laboratory (unpublished) have shown a relationship between delayed light emission (measured 3 msec after illumination, using a Becquerel phosphoroscope) and photosynthetic phosphorylation. Meanwhile, Jagendorf and Uribe5 have shown that synthesis of ATP can be induced in chloroplasts, without illumination, through a transition from an acidic to a basic environment. Therefore, it was natural to inquire whether an acid-base transition could bring about luminescence as well as phosphorylation. It will be shown here that an acid-base transition, performed in the manner described by Jagendorf and Uribe, causes spinach chloroplasts to emit a flash of light having the spectrum of chlorophyll fluorescence. The dependence of this chemiluminescence on a variety of conditions will be described. Materials and Methods.-Chloroplasts were prepared from spinach by the method of Good.6 Briefly, leaves were ground in a chilled mortar with sand in an aqueous medium containing 135 gm/liter sucrose, 2.5 gm/liter bovine serum albumin (Sigma fraction V), 0.05 M Tricine, and 0.01 M potassium chloride, adjusted to pH 7.8 with sodium hydroxide. After grinding, the mixture was filtered through glass wool and centrifuged for 5 min at 4600 X g. The chloroplasts were washed once by dispersing the pellet in grinding medium and recentrifuging at 4600 X g. The chloroplasts were then resuspended in grinding medium and stored in ice until used. The chlorophyll concentration was determined by the method of Arnon.' In a typical luminescence experiment the chloroplasts were exposed to three consecutive environments. The first ("Treatment I") consisted of 10 limoles Tricine and 7 /Lmoles KCl in 1.0 ml HO, with chloroplasts present at a concentration of 0.08-0.14 mg chlorophyll per ml. Transition to the second environment ("Treatment II") was effected by adding 0.1 ml of 0.1 M succinic acid. The pH was 4.4 at this stage. The final transition, producing a basic environment ("Treatment III"), involved the rapid addition of 1.0 ml of 0.1 M Tris, with a syringe. This transition yielded a final pH of about 8.6 and generated a flash of luminescence. Many variations of this protocol, and the consequences for luminescence, are described in Tables 1 and 2. The chemicals used in this investigation were obtained from the following sources: ADP, Tris, and L-malic acid, Sigma Chemical Co.; propionic and fumaric acids, Fischer Scientific Co.; methylamine, J. T. Baker Co.; Tricine, General Biochemicals. CCCP was a gift from Dr. P. G. Heytler, Central Research Department of the E. I. duPont de Nemours Co. to Dr. C. C. Black of this laboratory; DCMU was a gift from Dr. P. B. Sweetser, Central Research Department of the E. I. duPont de Nemours Co. to Dr. T. E. Brown of this laboratory; and desaspidin was a gift from Dr. H. Baltscheffsky to Dr. P. Boger of this laboratory.