NATURAL INHIBITORS OF THE HILL REACTION 277 



The leaves of small black locust seedlings {Rohinia) contain negligible 

 soluble tannin, and their chloroplasts possess high activity in vitro; 

 the leaves of mature black locust trees contain tannin which is ex- 

 tracted with the chloroplasts, which show low or negligible capacities 

 for the Hill reaction when isolated in neutral 0.5i1/ sucrose or O.OoM 

 phosphate. Tea leaf tannins are modified and degraded during tea 

 manufacture (22) ; extracts of commercial tea often are ineffective as 

 chloroplast inhibitors (2). When we collected Spirogijra from its 

 natural habitat, tannin was regularly present; when samples were held 

 for about one week in a laboratory aquarium provided with artificial 

 light, the tannin disappeared. Every sample of red clover leaves 

 (7". pratense) which we examined was rich in soluble tannins, and their 

 chloroplasts were consistently devoid of Hill reaction activity in 

 vitro; samples of white clover leaves {T. repens) collected simulta- 

 neously contained only traces of soluble tannin, and their chloro- 

 plasts were consistently active in vitro. 



Chloroplast inactivation by leaf tannins probably has the same 

 basis as the vegetable tanning of collagen, in which water of hydra- 

 tion is replaced by tannin (23). Although precise information is lack- 

 ing, it is apparent that the action of tannins on different enzymatic 

 systems differs greatly. So far as is known, the synthesis and degra- 

 dation of tannins is accomplished enzymatically — tannase and poly- 

 phenol oxidase are two plant enzymes w^hich must be quite insensitive 

 to tannin. Tannins usually are abundant in tobacco leaves, whose 

 chloroplasts possess only feeble activity in vitro (2) ; the fact that ac- 

 tive polyphenol oxidase, cytochrome oxidase, and catalase prepara- 

 tions have been obtained from tobacco leaves (24) suggests that these 

 enzymes are less sensitive to tannins than chloroplasts. Phosphatase 

 (25), amylase (25), hyaluronidase (26), as well as phages (27) and in- 

 fluenza virus (28), on the other hand, are inactivated by low tannin 

 concentrations. 



The intracellular location of tannins was studied histochemically 

 to learn why chloroplasts are photochemically active before but not 

 after cell rupture in their presence. The palisade parenchyma of hard 

 maple and sumac leaves contain tannin in their cell walls as well as in 

 the cell sap. Tannin granules also were detected in maple leaf cyto- 

 plasm. Special cells (idioblasts) which were completely filled with 

 tannin were also observed in these and other species. Tannin was con- 

 centrated in the end walls of the examined Spirogyra filaments. 



