Chapter IV —75— Nepenthes 



evident, due possibly to the inhibiting effect of the glycerine present. 

 These results were obtained on pitcher secretion preserved with 50% 

 glycerine, from N. Hibherdii and N. mixta. The secretion from open 

 pitchers containing insects, mostly ants, was used. In order to exclude 

 the effect of microbes and the enzymes of insect bodies, the authors 

 also took the glandular walls, comminuted and extracted them with acetic- 

 glycerine. The extract they found active on gelatine at pH 8, and on 

 ovalbumin only in the region of pH 3-3.5, thus supplying evidence that 

 a tryptic ferment is secreted by the glands of the Nepenthes pitcher. 

 In order to compare the enzymes of Nepenthes with those of animals 

 they made tests of the effect on them of certain activators, known to 

 affect other proteinases, \\ath negative results. Neither HCN, H2S or 

 cystein have any effect on the proteinase, nor does enterokinase on the 

 tryptase; the latter Stern had shown for the proteinase of white 

 blood cells. 



The conclusions of Stern and Stern, that there are two enzymes 

 present, a catheptic and a tryptic, and that the latter is not attribut- 

 able to the presence of bacteria, led W. de Kramer (1932) in Baas 

 Becking's laboratory at Leiden to re-examine the question. He came 

 to the conclusion that the opinion that the tryptic action is due to 

 bacteria is justified. De Zeeuw, who quotes de Kramer's unpubhshed 

 results, attacked this question. Both catheptic and tr>T>tic action was 

 found by them. De Zeeuw experimented with unopened pitchers 

 which were allowed to open under sterile conditions, using bromine 

 water and sterile cotton for insurance against bacterial infection, and 

 with unopened ones, which were always found sterile. 



The fluid of unopened pitchers does not digest fibrin until an acid 

 is added, an enzyme is therefore present. It becomes active within 

 the pH range of 3.4 to 4.4, phosphoric, malic and citric acid having 

 been used, and a phosphate buffer. That from an aseptically opened 

 pitcher acted at pH 3.6 in phosphoric acid, while that from normally 

 opened pitchers was active at pH 3.2 with phosphoric acid and from 

 7.2 and 8.6 with phosphate buffer. The last named was not sterile. 

 Bacterium fluorescens liquefaciens, B. prodigiosum and two others were 

 present, and all of these were found to exert tryptic action. By way of 

 control the fluid of a pitcher, opened under sterile conditions, of 

 N. Morganiana, was tested and found to digest fibrin at pH 4.4 to 5.5, 

 the pH increasing steadily during 15 days. An acetic acid-glycerine 

 extract was found to digest fibrin at pH 2.3 to 4.2, in direct contra- 

 diction to the results of Stern and Stern (1932) who also believed 

 their extract to be bacteria-free. 



Open pitchers display a wide range of pH (3.0-7.2), S3% reacting 

 neutral or basic, 36 pitchers being examined. When completely di- 

 gested insect cadavers were present, the fluid was neutral or weakly 

 basic; when digestion was in its early stages, acid. Into a pitcher 

 which showed an acid reaction (pH 3.0) the acid was neutralized by 

 means of hme water, and a pH of 8.2 established. Since digestion was 

 proceeding, the next morning the fluid was found to be at pH 3.0 again. 

 Pitchers after being washed out thoroughly with distilled water were 

 then supplied with distilled water (pH 7). When fibrin was added, the 

 pH dropped to 3.5, as also when egg-albumin (such as used by Clautriau) 



