THE DEMONSTRATION OF AN INDUCED, OR COWLED, REACTION JAMES B. SUMNER Biochernistv Labomcory, Cornell University,Ithaen, New York
DEMONSTRATIONS are sometimes difficult to conduct successfully and are not invariably illuminating or instructive even when successful. The coupled oxidation of carotene, which occurs when a trace of unsaturated fat, soap, or free fatty acid is peroxidized by lipoxidase,l, an enzyme abundant in soybean meal, makes a demonstration which is satisfactory in many respects. This demonstration illustrates: 1. 2. 3. 4. 5.
Enzyme action. The presence of double bonds in a fat. Peroxidation of a fat. An instance of an induced reaction. The destruction of carotene by oxidation.
The directions are as follows: Place 5 cc. of acetone-alcohol solution of crystalline carotene in a dry 250-cc. Erlenmeyer flask and 5 cc. in a second flask. To the first flask add a very small drop of cottonseed, hempseed, or raw linseed oil, or the respective soaps, or the free fatty acids. Mix, and then add to both flasks 100 cc. of water. Add 2 to 3 cc. of M/2 phosphate buffer of pH about 6.5. SUMNER AND DOUNCE. Eneymobgie, 7, 130 (1939). SUMNBR, 3. Bid. Chem., 134, 531 (1940)
' SUMNER AND
Now add 5 cc. of aqueous soybean meal extract and mix. The yellow color will disappear very rapidly from the flask to which the fat, soap, or fatty acid was added. The carotene in the second flask will undergo a slow spontaneous oxidation but some color will persist for as long as 24 hours. In this experiment lipoxidase transfers molecular oxygen to the double bonds of the unsaturated fat, forming peroxides. In the act of forming the peroxides the carotene is somehow oxidized. Fatty acids which are already peroxidized have very little action upon carotene. PREPARATION OF =AGENTS
Carotene Solution.-Dissolve about 80 mg. of crystalline carotene (90 per cent beta and 10 per cent alpha) in 200 cc. of acetone and400 cc. of alcohol. Phos#hate Buffer.-Mix 60 cc. of 2 / M Na2HP04 with 40 cc. of M/2 KH2P04. The solution will have a pH of about 6.5 when diluted. Lifiozidase Solution.-Mix 10 g. of defatted soybean meal with 100 cc. of water and centrifuge to clarify. The supernatant is employed. It will keep for a week or more if preserved with a few drops of toluene and kept in the ice chest.
