200 PHYSIOLOGICAL CHEMISTRY 



stand at room temperature for 24 hours. Strain the extract through cloth or 

 absorbent cotton and use the strained material in the following demonstration. 



2. Demonstration of Sucrase. To about 5 c.c. of a i per cent solution of 

 sucrose, in a test-tube, add about i c.c. of a 2 per cent sodium fluoride intes- 

 tinal extract, prepared as described above. Prepare a control tube in which the 

 intestinal extract is boiled before being added to the sugar solution. Place the 

 two tubes at 38C. for two hours. 1 Heat the mixture to boiling to coagulate the 

 protein material, filter, and test the filtrate by Fehling's test (see page 26). 

 The tube containing the boiled extract should give no response to Fehling's test, 

 whereas the tube containing the unboiled extract should reduce the Fehling's 

 solution. This reduction is due to the formation of invert sugar (see page 41 ) 

 from the sucrose through the action of the enzyme sucrase which is present in 

 the intestinal epithelium. 



For preparation and demonstration of Vegetable Sucrase see Chapter I. 



3. Preparation of an Extract of Lactase. Treat the finely divided epithelium 

 of the small intestine of a kitten, puppy, or pig embryo with about 3 volumes of a 

 2 per cent solution of sodium fluoride and permit the mixture to stand at room 

 temperature for 24 hours. Strain the extract through cloth or absorbent cotton 

 and use the strained material in the following demonstration. 



4. Demonstration of Lactase. 2 To about 5 c.c. of a i per cent solution of 

 lactose in a test-tube add about i c.c. of a toluol-water or a 2 per cent sodium 

 fluoride extract of the first part of the small intestine 3 of a kitten, puppy, or pig 

 embryo prepared as described above. Prepare a control tube in which the 

 intestinal extract is boiled before being added to the sugar solution. Place the 

 two tubes at 38C. for 24 hours. At the end of this period add i c.c. of the diges- 

 tion mixture to 5 c.c. of Barfoed's reagent 4 and place the tubes in a boiling water- 

 bath. 6 Examine the tubes at the end of three minutes against a black back- 

 ground in a good light. If no cuprous oxide is visible replace the tubes and 

 repeat the examination at the end of the fourth and fifth minutes. If no reduc- 

 tion is then observed permit the tubes to stand at room temperature for 5-10 

 minutes and examine again. 6 



It has been determined that disaccharide solutions will not reduce Barfoed's 

 reagent until after they have been heated for 9-10 minutes on a boiling water- 

 bath in contact with the reagent. 7 Therefore in the above test, if the tube con- 

 taining the unboiled extract exhibits any reduction after being heated as indicated, 

 for a period of five minutes or less, and the control tube containing boiled extract 

 shows no reduction, it may be concluded that lactase was present in the intestinal 

 extract. 8 



1 If a positive result is not obtained in this time permit the digestion to proceed for a 

 longer period. 



2 Roaf : Bio-Chemical Journal, 3, 182, 1908. 



3 Duodenum and first part of jejunum. 



4 To 4.5 grams of neutral crystallized copper acetate in 900 c.c. of water add 0.6 c.c. of 

 glacial acetic acid and make the total volume of the solution i liter. 



6 Care should be taken to see that the water in the bath reaches at least to the upper 

 level of the contents of the tubes. 



6 Sometimes the drawing of conclusions is facilitated by pouring the mixture from the 

 tube and examining the bottom of the tube for adherent cuprous oxide. 



7 The heating for 9-10 minutes is sufficient to transform the disaccharide into mono- 

 saccharide. 



8 The reduction would, of course, be due to the action of the glucose and galactose 

 which had been formed from the lactose through the action of the enzyme lactase. 



