626 



SCIENCE 



[N. a Vol. XXXIII. No. 851 



calcium carbonate and none whatever re- 

 mained in the supernatant liquid. Accord- 

 ingly, this precipitate was filtered off on a 

 Buchner funnel and allowed to dry over sul- 

 phuric acid. 



It is well knovm that tannin as such can not 

 exist to any considerable extent in the pres- 

 ence of proteid matter, since these two sub- 

 stances form a precipitate, but as it has been 

 shown repeatedly by the ferric chloride and 

 similar tests that a body existed in the plant 

 cells which gave these tests, the conclusion was 

 dravm that this body must be poly-atomic- 

 phenol that would not precipitate proteid mat- 

 ter. Accordingly, gallic acid was selected for 

 our following experiments. 



Therefore, a quintuple set of experiments 

 were then carried on, one set with tannin, a 

 second set with gallic acid, a third set with 

 gallic acid plus enzyme, a fourth set with 

 sodium gallate, and a fifth set with sodium 

 gallate plus enzyme. The experiments were 

 made by putting about 33 c.c. of liquid 

 medium in each of a number of 200 c.c. 

 Erlenmeyer flasks. The tannin, gallic acid 

 and sodium gallate were added to the medium 

 in the following proportions: .025, .05, .1, .2, 

 .4, .6, .8 and 1 per cent. The amount of 

 enzyme was constant throughout the two 

 series in which it was used. 



These experiments showed that the organ- 

 ism (Cunninghamiella echinulata) used made 

 its best growth in the check and in the gallic 

 acid, and the next best growth in the sodium 

 gallate. The gallic acid plus the enzyme, the 

 sodium gallate plus the enzyme, and the tan- 

 nic acid all showed a tendency to check the 

 growth in the lower percentages and to com- 

 pletely inhibit it in the higher percentages. 

 However, the results with the next organism 

 (Glomerella rufomaculans) were radically 

 different and led to further investigations. 

 In preparing the enzyme with calcium car- 

 bonate, as suggested by Appleman's work on 

 catalase, it has been shown above that the 

 precipitated calcium carbonate carried down 

 the catalase completely, but upon testing the 

 supernatant liquid with guiacum it was shown 

 that the presence of an oxidizing enzyme still 



existed in solution. However, upon testing 

 the precipitate with guiacum after dissolving 

 out the calcium carbonate and acidifying, 

 with acetic acid, it also showed that a con- 

 siderable portion of this oxidizing enzyme was 

 carried down by the calcium carbonate, and 

 upon drying this precipitate it was shown to 

 absolutely lose its activity. Thus in drying 

 the catalase precipitated by calcium carbon- 

 ate the portion of the oxidizing enzyme car- 

 ried down with it was killed, thus explaining 

 the marked difference, as shown above. It 

 should be stated here that in the first experi- 

 ment referred to above the calcium carbonate 

 precipitate was not completely dry, while in 

 the second it was quite dry. Thus in the first 

 case a considerable amount of the oxidizing 

 enzyme still existed and exerted its influence 

 on the transformation of the gallic acid into 

 the tannin-like body, while in the second case 

 the oxidizing enzyme had been destroyed by 

 the drying and no such action took place to 

 any considerable extent. 



It was now evident that we had two enzymes 

 instead of one, and that they could be com- 

 pletely separated from each other by their 

 properties, as stated above, but in such a large 

 proportion of the oxidizing enzyme carried 

 down by the calcium carbonate precipitate it 

 became evident that methods would have to be 

 devised for obtaining the oxidizing enzyme 

 from the supernatant liquid. Accordingly, 

 the supernatant liquid was drawn off and 

 treated by the general method for enzyme 

 precipitation, namely precipitation with alco- 

 hol (60 per cent.). This precipitate carried 

 practically all the enzyme down with it. This 

 was allowed to settle and finally collected on a 

 Buchner funnel. As it had been shown that 

 it could not be dried, it was now prepared for 

 use by suspension in water, and in this man- 

 ner used in the following experiments. The 

 ferric chloride and other tests on the plant 

 cell contents showed the presence of a poly- 

 atomic phenol, and it was evident that the 

 formation of the tannin-like body from the 

 poly-atomic-phenol could probably be carried 

 out in artificial solutions. However, it has 

 been previously shown by Bertrand in work- 



