Light Scattering Changes Associated with the Production of a Possible Intermediate in Photophosphorylation

Isolated chloroplasts from spinach (1, 2) and wheat (3, 4) are capable of a dark phosphorylation at the expense of an unknown reactive, labile substance or state formed in an earlier period of illumination.This substance, XB (I), is optimally stable at low pH, where it may accumulate in the light to the equivalent of 1 mole of adenosine triphosphate for 8 moles of chlorophyll.Such high yields require the presence during illumination of a cofactor catalyzing cyclic electron flow (2).The criteria of cofactor and low pH dependence have been used in attempts to associate certain other reversible phenomena observed in illuminated chloroplast suspensions with the formation and decay of X8.Two positive correlations were found ( 5) : (a) with a reversible rise in pH of the suspending medium and (b) with a turbidity change thought to reflect an altered conformation of t.he particles.Packer (6,7) and Itoh, Izawa, and Shibata (8) have shown that chloroplasts scatter more light after treatment with strong red light, and that the original low scatter state is restored during a subsequent dark period.The relation of these effects to electron flow and photophosphorylation at pH values between 7.5 and 8.0 was noted by Packer.Dilley and Vernon (9) extended these observations to the acid pH range and proposed that such changes are an indication of the presence of a high energy intermedia,te, probably XE.Our comparative data on the kinetics of light scattering and of X8 to be reported here will show that the absorbance changes are secondary effects of the high energy condition. EXPERIMENTAL PROCEDUREFresh turgid spinach was obtained from the market.Whole chloroplasts were prepared by maceration of the laminae in sucrose medium, as described previously (I).Resuspension for 15 min in Tris, 20 mm, pH 8.0, containing NaCI, 10 mM, effected hypotonic swelling of the particles, which were then collected by centrifugation at 1000 x g for 10 min, washed in this same medium, and finally resuspended in Tris, 5 mM; 3,3-dimethylglutarate, 5 InM; and NaCl, 40 mM (Tris-dimethylglutarate-NaCl buffer).This buffer mixture was present in all illuminated