NATURAL INHIBITORS OF THE HILL REACTION 275 



was observed by Bishop, Lumry, and Spikes (12) upon chloroplasts 

 stored at low temperatures. 



The studies summarized below involved several hundred experi- 

 ments whose main purposes were the identification of natural inhibi- 

 tors and the elucidation of the action of Carbowax upon chloroplast 

 preparations. Chloroplasts were isolated and washed at ± 1°C., 

 and were maintained at 0°C. until tested manometrically with 

 quinone at 10° C. (within 2 hours). The activity measurements were 

 made on dilute chloroplast suspensions subjected to saturating 

 orange-red light. Tannin analyses were made on aliquots of the super- 

 nate obtained in the first centrifuging of heat-coagulated chloroplast 

 suspensions, tannin being calculated from the KMn04 titer before and 

 after its complete removal with collagen. Chlorophyll was determined 

 according to :\IacKinney (13). Leaf moisture was determined by dry- 

 ing at 100°C. Acidity of the leaf sap was assessed by grinding the 

 leaves thoroughly with sand m two parts of distilled water and meas- 

 uring the pH. 



SAPONINS 



Positive hemolysis tests for saponins were shown by over half of 

 the leaf extracts examined in an extensive survey conducted by the 

 Eastern Regional Research Laboratory, Philadelphia (14). Triter- 

 penoid saponins were more commonly present than the steroidal type. 

 Both of these classes include numerous molecular species. Our studies 

 have indicated that the reversible inhibiting action of concentrated 

 alfalfa leaf sap upon the Hill reaction is caused by its triterpenoid 

 saponins. The steroidal saponin (sarsasaponin) of Yucca leaves was 

 without effect at concentrations up to 1%. Inhibition by saponins in 

 our experience has always been reversed by washing. In common with 

 detergents, their inhibiting action resembles that of urethanes. 

 Surface-active compounds presumably penetrate the lamellae at the 

 protein-lipid interphases, where they become oriented along with the 

 similarly polar chlorophylls and phospholipids. Transport of absorbed 

 light energy may then be blocked as a result of their incorporation in 

 the chlorophyll films (15). 



VACUOLAR ACIDITY 



When "active" chloroplasts are briefly immersed in leaf sap having 

 a pH below 4.0 (or in dilute mineral and organic acid solutions of the 



