DIGESTIVE ENZYMES IN POIKILOTHEEMAL. VEETEBRATES 
183 
DESCRIPTION OF METHODS 
The reaction in the esophagus, stomach, and intestine of various animals was 
determined approximately by means of indicator papers as soon as possible after 
killing the animal. 
For digestion studies the fresh alimentary tract was thorouglily washed and 
cleaned from traces of pancreatic tissue and mesentery. When the stomach con- 
tained substances in process of digestion, it was ligated at both ends to prevent 
passage of its contents into the esophagus and intestine while being cleaned. Then 
the tract was cut into the divisions to be studied, each division opened by a longi- 
tudinal slit, and the contents thoroughly washed out with the view of preventing 
any contamination of mucosa of one region with enzymes from other sources. 
After cleaning the mucosa or other digestive tissue was removed, weighed (moist), 
and triturated in a mortar with the aid of crushed glass when necessary. The finely 
ground tissue was made into a brei containing 10 per cent toluol, 40 per cent tissue 
for proteolytic enzymes or 25 per cent tissue for carbohydrate-splitting enzymes, 
with distilled water to 100 per cent. The brei was allowed to extract two to three 
days at room temperature and was strained through two thicknesses of cheesecloth. 
Coagulated egg white was used as substrate for pepsin and trypsin. It was 
prepared in considerable quantity and preserved in 10 per cent toluol. In making 
this substrate whites of eggs were repeatedly strained through several thicknesses 
of cheesecloth. To five parts of egg white four parts of water were added and the 
egg white coagulated by heat while being stirred constantly. One part of toluol 
was added and the mass stirred to uniform consistency. This made a very finely 
divided product, of 50 per cent egg white (moist weight), which could be readily 
pipetted. 
In the experiments on peptic and tryptic digestion the digests were made up 
to contain 12.5 c. c. of brei and 20 c. c. of coagulated egg white per 50 c. c. total. 
The approximate pH value of each digest was determined by paper indicators. 
For peptic digestion 0.2 N hydrochloric acid was added until the point was reached 
where congo red was just turned blue, a pH of about 3. In 50 c. c. digests about 
15 c. c. hydrocliloric acid were required. For tryptic and ereptic digestion tests 
0.2 N sodium carbonate was added until phenolphthalein gave the first pinkish tint, 
at a pH of about 8.4. In the alkaline digests 2 to 4 c. c. of toluol were added in 
addition to that contained in the brei and egg white to prevent any bacterial action. 
Casein was chosen as substrate for ereptic digestion, since this protein is readily 
split by erepsin, while most other native proteins, such as egg albumin, are not. 
In the ereptic digestion tests 2 grams of powdered casein were used per 50 c. c. 
digests. 
The progress of digestion was determined for pepsin, trypsin, and erepsin 
in terms of both the initial cleavage rate (tyrosine production), as shown by the 
colorimetric method of Folin and Denis (1912), and the amino acid production by 
the Sorenson formol titration method. The rate of digestion by the carbohydrate- 
splitting enzymes was determined in terms of the reducing power in milligrams 
of glucose per c. c. digest by Benedict's micro method for quantitative determina- 
tion of sugar. In studying the rate of starch digestion the iodine test was also 
employed. Careful controls were made by the use of boiled extracts. 
