water. In the great majority of instances where regeneration occurs, however, it cannot be due to any disturbance in the amount of water present in the parts concerned. Several leaves of Begonia and Bryophyllum were kept in the air of the room, but with their petioles in water. The blades quite plainly were not more turgid than those left on the plants, and not so much so as those on plants grown in moist air; yet they produced buds while the latter did not.

The results of experiments with the other factors mentioned will be presented in the second paper of this series.

THE UNIVERSITY OF CHICAGO.

LITERATURE CITED.

1. DECANDOLLE, A. P., Physiologie végétale. 2 vols. Paris. 1832.

2. GOEBEL, K., Ueber künstliche Vergrünung der Sporophylle von Onoclea Struthiopteris Hoffm. Ber. Deutsch. Bot. Gessells. 5:66-74. 1887.

3.- Ueber Regeneration im Pflanzenreich. Biol. Centralbl. 22:385

4.

397, etc. 1902.

-, Organography of plants, especially of the archegoniates and spermatophytes. Part I. pp. 270. figs. 130. Oxford. 1900.

5. KLEBS, G., Willkürliche Entwickelungsänderungen bei Pflanzen. Pp. 166. figs. 28. Jena. 1903.

6. KÜSTER, E., Beobachtungen über Regenerationserscheinungen an Pflanzen. Beih. Bot. Centrabl. 14:316–326. 1903. See also literature quoted in this paper.

7.

MORGAN, T. H., Regeneration. pp. 316. New York. 1901.

8. PFEFFER, W., Physiology of plants. Oxford. Vol. II. p. 167.

9. PRANTL, K., Untersuchungen über die Regenerationen des Vegetationspunktes an Angiospermenwurzeln. Arbeit. Bot. Inst. Würzburg 1:546, etc. 1874.

10. SACHS, J., Physiologische Untersuchungen über der Keimung der Schminkhohne. Gesam. Abhandl. Pflanzen-Physiologie Abh. 25:574.

II. SIMONS, S., Untersuchungen über die Regenerationen der Wurzelspitze Jahrb. Wiss. Bot. 40:103-143. 1904.

12. VÖCHTING, H., Ueber die Regeneration der Marchantieen. Jahrb. Wiss. Bot. 16:316-414. 1885.

13.

Ueber Organbildung im Pflanzenreich. Bd. I. Bonn. 1878.

14. WAKKER, J. H., Onderzoekingen over adventiene Knoppen. Acad. Proefschrift. Amsterdam. 1885.

15. WIESNER, J., Der absteigende Wasserstrom und dessen physiologische Bedeutung. Bot. Zeit. 47:1-9, etc. 1889.

ON PROTEOLYTIC ENZYMES. II.

ARTHUR L. DEAN.

SINCE the writing of the previous paper1 on the vegetable proteolytic enzymes, an article by VERNON' has appeared in which he gives an account of his investigation of the distribution of erepsin in the animal body. Comparative tests were made of glycerin extracts from thirteen different tissues of the cat, eight of the rabbit, eight of the guinea pig, seven of the pigeon, eight of the frog, seven of the eel, six of the lobster, and three of the fresh water mussel. In every case an erepsin was found to be present. The comparison of the activity of the various extracts was effected by means of colorimetric estimations of the intensity of the biuret reaction after certain periods of digestion. The tissue extracts from the warm blooded animals were more active than those from the cold blooded animals; the extracts from the invertebrate tissues had a relatively slight action. In the warm blooded animals it is not the intestinal mucous membrane which is richest in erepsin, but the kidney in the cat, rabbit, and pigeon, and the pancreas in the case of the guinea pig.

It might be noted that VERNON gives no record of experiments made with the various extracts to determine whether or not they are incapable of digesting the prot ids of the tissues in which the enzymes occur. His experiments, being all conducted with Witte peptone solutions, do not conclusively show that the enzymes whose activity was observed are incapable of acting on any proteids except albumoses and peptones.

In a former communication evidence was given to show that Phaseolus vulgaris contains in its seeds a fairly active proteolytic enzyme. No action of this protease on the proteids of the seed could be demonstrated. Its power to act on Witte peptone as a whole, and on the albumose fractions separated from it, could be readily shown. Moreover, as germination progressed the cotyledons at all stages contained this ereptase and at no period of germination could 1 DEAN: BOT. GAZETTE 39:321. 1904.

any evidence of a tryptic enzyme be obtained. Further studies have been made of this seed enzyme in the hope of throwing light on the processes occurring during germination.

FURTHER EXPERIMENTS WITH THE GERMINATING SEEDS OF PHASEOLUS VULGARIS

The following experiment was carried out to demonstrate the action of the seed ereptase of the bean on the proteoses to be obtained from phaseolin, the principal globulin of the seed. Phaseolin was prepared according to OSBORNE'S3 method from white medium field beans, the yield from two kilograms of beans amounting to something over 758m. A small amount of phaselin was also obtined by the alcoholic precipitation of a part of the solution from which the phaseolin had separated on dialysis. This albumin gives a marked Adamkiewicz reaction.

By mixing a known weight of phaseolin with fifty times its weight of 2 per cent. sulphuric acid and heating for three and a half to four hours in a steam sterilizer, a slightly brown gelatinous fluid was obtained having the odor characteristic of acid proteid hydrolysis. This fluid was cooled, diluted with water, and the gelatinous antialbumid removed by filtration. The filtrate from the antialbumid was neutralized with barium hydroxide and the filtrate from the barium sulphate concentrated and treated with sev ral volumes of alcohol. The mixture of proteoses obtained in this way was collected on a filter, washed with absolute alcohol and ether, and dried in vacuo over sulphuric acid.

To obtain an ereptase solution finely ground beans were extracted with water and the extract filtered through pulp filters until nearly clear. Ten cubic centimeters of this solution were placed in each of four dry test tubes and two of the portions boiled. Solutions of Witte peptone and phascolin proteoses were prepared by dissolving 0.28m of each preparation in 25cc of water. The test solutions were made up as follows:

I. 10cc enzyme solution + 10°c phaseolin proteose solution +9 drops of toluol. 2. Same as no. 1, using boiled enzyme solution.

3. 10 enzyme solution +10° Witte peptone solution +9 drops of toluol.

4. Same as no. 3, using boiled enzyme solution.

3 OSBORNE: 17th Annual Report of the Conn. Agric. Experiment Sta., Part IV. 186. 1893.

CC

The corked tubes were kept in the incubator for six days. At the expiration of that time each digestion was boiled, acidified with a couple of drops of acetic acid, and filtered. Ten cubic centimeters of each filtrate were removed to a clean tube, and, after the addition of 5cc of 10 per cent. sodium hydroxide solution, dilute copper sulphate solution was added to the maximum biuret reaction. After standing for several minutes the intensity of the biruet reactions was compared. The comparison showed that a marked digestion of both proteose mixtures had taken place, the one from phaseolin being rather more vigorously attacked. The unboiled digestion with Witte peptone gave a tryptophan reaction with bromine water, the other unboiled digestion did not.

Several trials were made of the digestibility of the acid phaseolin prepared by heating phaseolin for a few minutes with dilute sulphuric acid. The results obtained indicated that the enzyme of the seed had, at the most, but a feeble action on this body.

Various observers have shown that the antiseptics used in enzyme experiments may exert an inhibiting effect on the action of the ferment. VINES demonstrated that in papain digestions where sodium fluoride is used the action goes but little beyond the stage where albumoses and peptones are formed; whereas with other antiseptics, hydrocyanic acid for example, a marked formation of amido-acids takes place as shown by the production of tryptophan. It might be argued that the toluol used throughout the experiments with Phaseolus had an inhibiting action on the enzyme, so that it was unable to attack the proteids of the seed and could only act on albumoses and peptones. The two following experiments were carried out to settle that point:

Experiment I.--Thirty-five grams of finely comminuted cotyledons from six-day old bean seedlings were extracted for one and a half hours with 175° of water. The extract was filtered nearly clear through pulp filters and then forced through a PasteurChamberland filter into a sterile flask. Three portions of 25cc each were measured with a sterile pipette into three small sterile flasks. To no. I nothing further was added; to no. 2 was added 0.07 gm N 25

of sterile citric acid, making the solution citric acid; the contents

of no. 3 were boiled. The cotton plugs in the flasks were replaced corks. After keeping the three flasks in the incubator for forty-one hours, 20°c of tannic acid reagent (7 per cent. tannic acid in 2 per cent. acetic acid) were run into each. After standing a few moments the contents were filtered through dry filters and duplicates of 20 cc from each filtrate analyzed for nitrogen by the Kjeldahl method.

Analyses of filtrates from no. I gave 0.00368m N
Analyses of filtrates from no. 2 gave 0.00368m N
Analyses of filtrates from no. 3 gave 0.00338m N

Experiment II.-Another extract from cotyledons of six-day old seedlings was prepared by treating 288m of finely ground tissue with 140cc of cold water for three hours. The extract was filtered as in the previous experiment and 25cc of the filtrate removed into two sterile flasks. The contents of one flask were boiled and both were corked with sterile corks and kept in the incubator for three days. To test the ereptic activity of the bacteria-free filtrate 15cc were placed in a test tube with 0.5gm of Witte peptone and a little toluol. At the end of the digestion period the fluid in this tube was found to give a strong tryptophan reaction with bromine water, showing that the enzyme had not been held back by filtration through porcelain. The contents of the two flasks were treated as in the previous experiment:

Analyses of filtrate from unboiled digestion gave 0.00368m N.
Analyses of filtrate from boiled digestion gave 0.0030 gm N.

The differences between the boiled and unboiled digestions in these two experiments are so slight as to be within the limits of error of the method used and cannot be taken to show any hydrolysis of the proteid in the cotyledon extract.

PROTEOLYSIS DURING THE GERMINATION OF PHASEOLUS VULGARIS

We have every reason for believing that the germination of the bean is accompanied by a hydrolysis of the proteids therein contained, a hydrolysis which our experimental evidence leads us to conclude must be started, at least, by some other agency than an enzyme. That proteolysis does accompany germination of this seed is shown by the following experiments, where the amounts of