Exercise V 



ENZYMES 29 



(7) If the activity of the saliva is not too great 

 (if, for example, it takes more than a minute 

 for the 1:19 dilution to reach the endpoint), 

 the remaining two dilutions (1 :49 and 1 :99) can 

 be run simultaneously to save time, and the test 

 intervals can be increased again to keep pace 

 with the rates of reaction. 



Plot a graph showing the reciprocal of the 

 time (1/min) required to reach the endpoint vs. 

 the concentration of saliva. The reciprocal of 

 the time is a measure of the rate of reaction. 



Compare the activity of the saliva used in 

 your tests with that used by other students in 

 terms of the times required to reach the achro- 

 matic endpoint in the tubes to which 2% saliva 

 was added. Compare the result in your own 

 experiment with the minimum, maximum, and 

 mean values of the class as a whole. What 

 would you conclude? 



Reaction rate vs. temperature. (Note: Half 

 the students in the class should do this experi- 

 ment, the other half the experiment on acidity 

 below, in each case working in pairs.) Using 

 the same techniques and the same pH as in the 

 previous experiment, and selecting a saliva con- 

 centration that yields an endpoint in 3 to 4 

 minutes, determine the rate of the reaction at 

 0°C (ice in water), room temperature, 37°C 

 (water bath), and 100°C (boiling water). At 0° 

 and 100°, tests can be made at intervals of 1, 5, 

 or 10 minutes, after it has become clear that 

 the reaction is going slowly. 



Plot a graph of 1/min to endpoint vs. tem- 

 perature. 



Chemical reactions in general go 2 to 3 times 

 faster for every 10° rise in temperature. The 

 same tends to be true of enzyme-catalyzed reac- 

 tions, with a special twist: as the temperature 

 rises, it reaches a point at which it begins to 

 destroy the enzyme, as it does other proteins, 

 and thereafter the reaction rate falls instead of 

 rising further. The result is that as the tem- 

 perature is raised from some low initial value, 

 the rate of the catalyzed reaction first rises, then 

 falls. At a certain temperature, just before it 

 begins to fall, the rate is at its highest, the 

 so-called temperature optimum. 



From your observations, about where do you 

 estimate the temperature optimum for salivary 

 amylase to lie? How is it related to your body 

 temperature? If you now brought both the 0° 

 and the 100° samples to 37°, what reaction rates 

 would result? Why? 



Reaction rate vs. pH. Determine the time to 

 reach the endpoint at 37° in reaction mixtures 

 buffered at pH 3.4, 5.0, 6.8, and 8.0. Mix 2 ml 

 of the starch-NaCl solution, 2 ml of the appro- 

 priate buffer, and 1 ml of a dilution of saliva 

 deemed suitable on the basis of your previous 

 measurements. 



What do you conclude to be the approximate 

 pH optimum of salivary amylase? On what 

 side of neutrality does it lie? How is it related 

 to the pH of your saliva? (Measure this by 

 touching the end of a piece of pHydrion paper 

 to your tongue and comparing with the color 

 scale.) 



Whichever of the last two experiments you 

 did, find out what results were obtained in the 

 other experiment, and note them in your labora- 

 tory notebook. In general we want you to know 

 everything that goes on in your laboratory, 

 whether you do it yourself or not. 



PHOSPHORYLASE 



For a long time it was thought that such 

 amylases as you have just examined are responsi- 

 ble for degrading glycogen in animal tissues. Yet 

 liver and muscle degrade glycogen very much 

 more quickly than any known amylases can 

 accomplish. In 1935 a new class of polysac- 

 charide-splitting and -synthesizing enzymes was 

 discovered, called phosphorylases. The splitting 

 of glycogen by a phosphorylase requires the 

 presence of inorganic phosphate, and the prod- 

 uct is not glucose, but glucose- 1 -phosphate. 

 Whereas amylases break glycolytic linkages by 

 introducing water (hydrolysis), phosphorylases 

 do the same job by introducing phosphoric acid 

 (phosphorolysis), as shown on the next page. 



