Exercise V 



ENZYMES 27 



Try to tind time after doing the experiments to 

 make molecular models of succinic and malonic 

 acids. What resemblance between these mole- 

 cules do you suppose fools the enzyme? 



Experiment 



Succinic dehydrogenase occurs in the cell 

 particles known as mitochondria. It can be ob- 

 tained directly from beef heart. A piece of meat 

 about the size of a small marble should be used 

 for each assay. The meat should be cut up 

 further and washed a few times by vigorous 

 shaking with water in a test tube, followed by 

 decantation of the wash water in order to re- 

 move any substrates already present. Add re- 

 agents to the labeled tubes as follows, agitating 

 so that added substances are evenly distributed 

 throughout the muscle suspension: 



Tube 1: no meat; add an equivalent volume of 

 water plus 3 drops succinic acid (0.5 M) 

 and 7 drops methylene blue (MB) solu- 

 tion (0.01%). 



Tube 2: meat plus 3 drops succinic acid. 



Tube 3: meat plus 7 drops MB. 



Tube 4: meat plus 3 drops succinic acid plus 7 

 drops MB. 



Tube 5: meat plus 3 drops succinic acid plus 3 

 drops malonic acid (1 M) plus 7 drops 

 MB. 



Tube 6: meat plus 9 drops succinic acid plus 3 

 drops malonic acid plus 7 drops MB. 



Tube 7: boiled (2 min) meat plus 3 drops suc- 

 cinic acid plus 7 drops MB. 



Bring the solutions in all tubes to the same 

 total volume (19 drops, as in Tube 6) by adding 

 distilled water. 



Pour mineral oil down the side of each tube 

 so as to form a surface layer not more than 1 cm 

 thick. The oil keeps oxygen from diffusing in, 

 and so prevents reoxidation of MB-H2. Place 

 the tubes in the water bath at 37°C, and watch 

 for color changes while you go on with other 

 experiments. 



What changes have you observed? Why was 

 the experiment set up in seven test tubes as 

 above? What does each mixture contribute? 

 Could you have learned as much from fewer 

 mixtures? At the end of the experiment, you 

 can demonstrate the rapid oxidation of re- 

 duced methylene blue by air by stoppering 

 Tube 4 with your finger and shaking it vio- 

 lently. 



SALIVARY AMYLASE 



Starches are very large carbohydrate mole- 

 cules, made by stringing hundreds to thousands 

 of glucose molecules together in long straight 

 and branched chains (Review Exercise III). 

 Saliva, which is secreted by the salivary glands in 

 amounts of the order of 1 liter daily, contains an 

 enzyme that catalyzes the hydrolysis (digestion) 

 of starch through a series of smaller and smaller 

 intermediates (so-called dextrins) to the final 

 product maltose, which consists of two glucose 

 molecules joined together as in starch. To hy- 

 drolyze maltose to glucose requires another 

 enzyme, maltase, not present in saliva, but 

 secreted by both the pancreas and the small 

 intestine. 



The salivary enzyme that hydrolyzes starch to 

 maltose is called salivary amylase. It has been 

 prepared in crystalline condition. Older names 

 for it are salivary diastase and ptyalin. 



Its action can be followed readily with the 

 iodine test. Iodine yields a deep blue color with 

 starch (actually only with the straight-chain, 

 amylose fraction of starch). As the starch is hy- 

 drolyzed, repeated tests with iodine go from the 

 initial blue color to red or reddish brown (dex- 

 trins), and eventually to colorlessness (smaller 

 dextrins, maltose). 



A word about the shapes of molecules and 

 specificity of enzymes. Like starch, cellulose is 

 made of glucose molecules tied together to form 

 very long chains. The only essential difference 

 between starch and cellulose — one of the most 

 biochemically reactive, and one of the most 

 inert molecules — is that in starch the glucose 



