NATURAL INHIBITORS OF THE HILL REACTION 279 



CARBOWAX-TANNIN INTERACTION 



The first indication of Carbowax-tannin interaction was obtained 

 when the collagen method for tannins was applied to leaf extracts 

 containing 30% Carbowax 4000. Tannins are readily removed by col- 

 lagen from leaf extracts prepared with 0.5 M sucrose or 0.05 M phos- 

 phate, but they are not removed completely from Carbowax extracts 

 even when the amount of collagen is trebled. Further experiments 

 showed that Carbowax also interferes in the adsorption of tannin by 

 chloroplasts. The foregoing phenomena apparently are caused by the 

 affinity of Carbowax for tannin in solution : leaf tannins are removed 

 from simple ac^ueous solutions by liciuid-liquid extraction with ethyl 

 acetate, but under otherwise similar conditions they are not ex- 

 tracted by ethyl acetate from 30% Carbowax 4000 solutions. The 

 affinity of Carbowax for tannins m solution probably was also re- 

 sponsible for its frequently observed inhibition of the enzymatic 

 browning reaction in leaf macerates (which results from the action of 

 polyphenol oxidase upon " tannins" (29)). 



Because of the above effects of Carbowax upon tannins, one might 

 expect to obtain active chloroplasts from certain species with Carbo- 

 wax, though the same species yield essentially inactive chloroplasts 

 when ground in sucrose solutions, etc. Several examples of this fa- 

 vorable effect of Carbowax were encountered (walnut, red oak, hard 

 maple, black locust, and geranium leaves). Examples also were en- 

 countered, however, of leaves which contained sufficient soluble tan- 

 nin for chloroplast inactivation when 30% Carbowax 4000 was em- 

 ployed as grinding medium at 0°C. (sumac, red clover, ragweed 

 leaves) . 



Chloroplasts which exhibit negligible Hill reaction acti\aty after 

 mild w^ashing rarely show improvement upon exhaustive washing, but 

 this is a characteristic of red elm chloroplast suspensions. When 

 these chloroplasts were isolated at 0°C. in accordance with Mc- 

 Clendon (4), they exhibited oxygen absorption in light when neutral 

 30% Carbowax 4000, 0.5 M sucrose, and 0.05 M phosphate were em- 

 ployed as grinding and dispersion media. The supernates did not in- 

 hibit active chloroplasts, and the soluble tannin content was no 

 higher than in several "active" species; the leaf sap also was approx- 

 imately neutral. After four washes with 30% Carbowax 4000, the 

 red elm chloroplasts which previously were "inactive" now exhibited 



