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



same pH), Hill reaction activity is lost irreversibly. This effect is 

 eliminated, in the absence of other inhibitors, when the foreign leaf 

 sap (e.g., Oxalis) is neutralized beforehand. Acid inactivation occurs so 

 rapidly that chloroplasts isolated from acidic leaves possess no Hill 

 reaction activity when the grinding fluid is strongly buffered with 

 phosphate at neutrality — the plastids are subjected to the vacuolar 

 sap during the homogenization which precedes release of the cell con- 

 tents. Acid inactivation probal)ly could be eliminated by soaking the 

 leaves in dilute ammonia prior to maceration (16). This common tj^pe 

 of chloroplast inactivation is unaffected by the use of concentrated 

 Carbowax solutions. 



Extensive information on the fluctuating acidities of leaf sap has 

 been summarized by Small (17). The expressed sap of Oxalis, Begonia, 

 and Opuntia leaves may have pH values as low as 1.4 when the leaves 

 are collected early in the morning ; by late afternoon the pH may rise 

 to 5.5 in the same leaves. The sap of conifer needles (17) and of 

 Gingko leaves usually has a pH below 4.0. Eight families of "acid" seed 

 plants are listed by Small (17). The acidity gradient within the pine- 

 apple leaf observed by Sideris and Young (18) in itself would result in 

 the inactivation of chloroplasts isolated from the apex (pH 3.3) but 

 not from the base (pH 5.5) of the same leaf. 



TANNINS 



Tannins are common constituents of leaves (9) as well as of multi- 

 cellular marine (19) and fresh-water algae (20). There are many differ- 

 ent kinds of tannins, and the tannin from any one plant is often a mix- 

 ture of poly phenolic compounds. Thus twenty-seven different poly- 

 phenols were obtained by countercurrent fractionation of Acacia ex- 

 tract (21). Identification of tannin as a chloroplast inactivator is 

 readily accomplished with collagen: neutral aqueous extracts of cer- 

 tain leaves (e.g., Rhus fyphina) which abolish the Hill reaction ir- 

 reversibly in "active" chloroplasts lose this effect when the tannins 

 are removed completely by selective adsorption on collagen. 



Some evidence has been obtained of the variable effectiveness of 

 different tannins as chloroplast inhibitors, and of varying contents of 

 tannin within the same as well as related species. The leaves of mature 

 honey locust trees (Gleditsia) regularly give positive histochemical 

 tests for tannin; during chloroplast isolation, Gleditsia tannin re- 

 mains insoluble and the chloroplasts possess high activity in vitro. 



