1 6 PHYSIOLOGICAL CHEMISTRY 



peroxide as well as certain biological peroxides with the liberation of 

 oxygen. Catalase is an indirect oxidizing enzyme, that is, it brings about 

 oxidations only in the presence of a peroxide. Catalase is also found in 

 many plant tissues and an extract of it may be prepared from potatoes. 



1. Vegetable Catalase. Into each of four test-tubes place 5 c.c. of filtered 

 potato extract prepared as in Experiment 2, page 14. Prepare a second 

 series of four tubes (see 4, p. 15), but use a boiled potato extract. Prepare 

 also a third series using water instead of potato extract. Now add to each 

 of the twelve tubes 5 drops of a 3 per cent solution of hydrogen peroxide. While 

 the resultant lively effervescence is in progress add to each series the four 

 "Typical Oxidase Reagents" hi the order and quantities specified in the preceding 

 experiment^). Allow the tubes to remain undisturbed and carefully note com- 

 parative effects during the remainder of the laboratory exercise. 1 



2. Annual Catalase. The presence of this enzyme may also be demonstrated 

 as follows: Introduce into a low, broad, wide-mouthed bottle some pulped liver 

 tissue and a porcelain crucible containing neutral hydrogen peroxide. 2 Connect the 

 bottle with a eudiometer filled with water, upset the crucible of hydrogen peroxide 

 upon the liver pulp and note the collection of gas in the eudiometer. This gas is 

 oxygen which has t>een liberated from the hydrogen peroxide through the action of 

 the catalase of the liver tissue. 



See next experiment for a method for the quantitative determination of catalase 

 based on the above principle. 



3. Quantitative Determination of Catalase. 3 In the determination of the 

 catalase values of tissues weighed portions of the tissue under examination should 

 be ground with sand in a mortar then treated with four volumes of chloroform water 

 and permitted to extract for 24 hours at room temperature. An apparatus such 

 as that shown in Fig. i may be employed in determining the catalase values. The 

 main features of the apparatus are based upon those of a delivery funnel for intro- 

 ducing liquids under increased or diminished pressure. 



In making a determination introduce a measured volume (1-4 c.c.) of the filtered 

 extract 4 into the small flask and insert the modified Johnson burette graduated to 

 5 c.c. and containing 50 c.c. of hydrogen peroxide (Oakland dioxygen neutral 5 to 

 Congo red) into the neck of the flask. Fill the eudiometer with water and place in 

 position. Close cocks A and C and open cocks B and D thus permitting 5 c.c. of 

 the peroxide to flow into the flask. Shake the contents of the flask briskly 6 and 

 record the volume of oxygen evolved in a two-minute period taking readings at 

 intervals of fifteen seconds. 



Calculation. When a series of comparative tests are made on different tissues 

 or on the same tissue under different conditions it is considered satisfactory to make 

 a comparison of the catalase values upon the basis of the volume of oxygen evolved 



1 This experiment has been adapted from one contained in the Laboratory Notes of 

 Professor Gies of the College of Physicians and Surgeons, New York. 



2 Mendel and Leavenworth : American Journal of Physiology, 21, 85, 1908. 



3 Hawk: Journal of American Chemical Society, 33, 425, 1911. 



4 If less than 4 c.c. of extract are used the volume should be made up to 4 c.c. by the 

 addition of distilled water. 



6 An acid reaction modifies the rate of the oxygen evolution. (See Mendel and Leaven - 

 worth, American Journal of Physiology, 21, 85, 1908.) 



6 In making a series of comparative tests it is essential that the shaking process should be 

 uniform from determination to determination. 



