Emerson Enhancement Effect in Chloroplast Reactions

Abstract

The discovery of the Emerson enhancement effect in photosynthesis (3-6, 8) led to the suggestion that there are 2 photochemical reactions necessary for complete photosynthesis. A likely hypothesis is that the 2 reactions are carried on by different forms of chlorophyll a (10, 11). The enhancement effect in photosynthesis has been studied extensively in sev-eral laboratories (19) and it has been shown (12, 14, 15) that the effect also occurs ill 02 evolution in the Hill reaction using quinone as the electron acceptor. (iovindjee et al. (13) have recently reported the ex-istence of the Emerson enhancement effect in NADP photoreduction by spinach chloroplasts using white (fluorescent) supplementary light. This paper deals w\vith observations on the enhancement of NADP lphotore(luction and 0., evolution in chloroplast prep-arationss, using both white and monochromatic lights. Materials and Methods Preparation of Chloroplasts. Chloroplasts from sl)inach leaves were prepared by a method similar to that of Hill and Walker (16). Fresh spinach leaves w\\ere first cooled in ice cold water, deribbed, and then chopped into small pieces. The chopped spinach leaves were ground with white sand using a mini-MuM quantity of a solution containing 0.01 At NaCI, 0.4 al sucrose, and 0.05 Mr Tris, pH 7.5. The result-ing susl)ension was filtered through 4 layers of cheese cloth and then centrifuged for 1 minute at 200 g. The cell debris was discarded and the supernatant fluid centrifuged for 10 minutes at 1000 to 1500 X g. This supernatant material was (liscar(le(l and the pel-let was suspended in buffer and recentrifuged at the same speed for 10 minutes. The supernatant solu-tion was again discarded and the chloroplasts made up in a known volume of the buffer. Chlorophyll concentration was estimate(l spectroscopically (1). Reaction Mixture. The reaction mixture used in the experiments had the following constituents in ALmioles per 2 ml of total volume: KH2PO 100 (pH 7.3); MgCl.,, 15 ; ADI3, 2; NADP, 1; chloroplasts containing 100 ug of chloroplhll. an(l an optimal l Received April 29, 1963. 2 This work was supported by the National Science Foundation (G 19437), Air Force Office of Scientific Research (Contract AF49 (638)-947) and the National Institutes of Health (RG-6692). 3 Postdoctoral Biophysics trainee of the United States Public Health Service. amount of photosynthetic phosphopyridine nucleotide reductase (PPNR). PPNR was prepared according to the method of San Pietro and Lang (20) and was partially purified through the Dowex-Bentonite step. Optical Svstemz. One beam of light was obtained from a 750 w tungsten lamp controlled by a variac. The light beam was made parallel. passed through a 30 cm water filter and appropriate interference and colored glass filters and was then focussed on a water-cooled Beckman cuvette that contained the reaction mixture. The maxima of the Bausch and Lomb sec-oncl order intererfence filters (35 % peak transmis-sion, half-band width 10 mu) and the numbers of their associated colored glasses are: 678 nmy (2-61 Corning), 693 mIu (RG-5 Schott and Gen.), 700 nm, (RG-5 Schott and Gen.), 714 mA (RG-8 Schott and Gen.), 721 ins (RG-8 Schott and Gen.), 730 mn1A (RG-8 Schott and Gen.), and 740 myA (RG-8 Schott and Gen.). A second beam of light, the supplementary white light, xvas obtained from a 40 Iv white fluorescent tube. This light (lid not contain any far-red wave-lengths. It was chosen so as to get a mixture of wavelengths that would excite both chlorophyll b