278 K. A. CLENDENNING, T. E. BROWN, E. E. WALLDOV 



Detectable amounts of tannin were found in the upper epidermal cells 

 of Phytolacca leaves, which are excellent chloroplast sources. Spinach 

 leavTs were consistently free of tannins and the pH of their leaf sap 

 was always abo^'e G.O. The fact that tannin occurs within specialized 

 photosynthetic cells (e.g., sumac, maple) is believed to establish that 

 chloroplast inactivation by tannin as well as acids can occur by the 

 homogenization of individual cell contents. Tannin is also contrib- 

 uted by nonphotosynthetic cells, and tannin which is located in the 

 cell walls is brought into solution during chloroplast isolation. Here 

 the temperature at which the chloroplasts are isolated is important — 

 under otherwise standard conditions, the amount of tannin extracted 

 with the chloroplasts usually increases when the maceration tempera- 

 ture is raised. 



Having shown that the inactivation of chloroplasts which occurs 

 during their extraction from many species is caused by tannins and 

 acids, we attempted to devise a procedure which would yield active 

 chloroplasts from acidic leaves of high tannin content. Sumac leaflets 

 were used in these studies because of their acidity (pH 4.0 to 4.2) and 

 exceptionally high tannin content. All of the extraction methods 

 which we tested failed to yield sumac chloroplasts with measurable 

 activity when the leaflets were macerated at 0°C. (large volumes of 

 grinding fluid with and without Carbowax and sucrose, buffered at 

 difl'erent pH values up to 9.0, and containing tannin adsorbents in 

 excess (collagen, egg albumen); vacuum infiltration of the intact 

 leaflets with bufl"ered egg albumen solutions). Photochemically active 

 sumac chloroplasts were eventually obtained by a very laborious 

 method, the Hill reaction proceeding at a low but steady rate for 1 

 hour at 10°C. To obtain these, 5-g. intact leaflets were cooled to 

 — 70°C. in a medium consisting of 35 ml. fresh egg white diluted to 

 100 ml. with 0.2 M phosphate, pH 7.0, containing 0.2 M sucrose, 0.1 

 M Carbowax 4000, and 0.01 AT KCl. The resulting cake was chipped 

 and then ground to a fine powder at ca. — 70°C. in the presence of 

 excess dry ice. The melted brei was processed in the usual way, em- 

 ploying large volumes of the foregoing medium as wash liquid. The 

 fact that photochemically active sumac chloroplasts were obtained 

 when the leaflets were ground at — 70°C. but not at 0°C. in the same 

 maceration medium strongly indicates that chloroplast inactivation 

 in this leaf is an intracellular phenomenon. 



