542 



ILLUSTRATIVE EXPERIMENTS 



45. The Influence of the Hydrogen Ion Concentration on the Activity of 

 an Enzyme. Ptyalin (adapted from Ringer, Zeits.f. physiol. Chem., 1910). 



Material required. (1) About 50 c.c. of a 1 in 50 dilution of saliva. (2) Seven 

 large boiling tubes or small Erlenmeyer flasks of about 50-100 c.c. capacity. 

 The tubes should contain phosphate buffers made up as on p. 525, so that 

 each tube has 5 c.c. of a phosphate solution with the following pK values : 

 6-24, 6-47, 6-64, 6-81, 6-98, 7-17, 7-38. (3) 0-5 per cent, solution of boiled 

 starch made up in 0-3 per cent, sodium chloride solution. (Ptyalin operates 

 best in the presence of the CI ion.) (4) A series of about 30 small tubes, each 

 containing 5 c.c. of an approximately N/1,000 iodine solution to act as 

 indicators. 



Method. To each boiling tube in turn add 5 c.c. of the diluted saliva and 

 mix. Starting from the left and at an interval of exactly two ininutes between 

 each flask, add 25 c.c. (or 50 c.c. if your tubes will contain it) of the starch sol. 

 Then at tAVO-minute intervals, 2 c.c. of tube 3 are transferred to an iodine 

 tube. At first the colour will be blue, later violet, later still red. At this 

 stage, without delay, remove in turn 5 c.c. of the contents of each tube and 

 transfer to separate iodine tubes. Note that as before, exactly two minutes 

 should elapse between the withdrawal of the reaction mixture from successive 

 tubes. The following is a typical resvdt : 



Tube Xo. 



_pH (approx.) 

 Iodine colour 



7-2 

 red violet 



7-4 

 violet 



In tube 4 the reaction has proceeded most rapidly, i.e. fR 6-8 (approx.) is 

 the optimum jpH for ptyalin. 



46. Effect of Removal of the End-products on the Rate of Action of 

 Ptyalin (p. 125). 



Place a small quantity of saliva in a test tube and dilute w^ith an equal 

 volume of water. Divide this amount equally between a dialysing cylinder 

 A and a slide-tube B of approximately the same diameter. Add an equal 

 quantity of 0-5 per cent, boiled starch (in 0-3 per cent. NaCl) to each tube 

 and mix the contents. Place the tubes in a beaker and maintain a 

 constant flow^ of water in the beaker. The flow of water will maintain a 

 comparatively steady temperature in both tubes and will hasten dialysis 

 in tube A. By means of a glass rod (one to be kept for each tube) transfer, 

 from time to time, a drop from each solution to a white glazed tile and add 

 to each drop a little iodine solution. In the drops from tube A a blue colour 

 with iodine is given at first ; later drops give a purplish, later still a reddish 

 brown colour, and after about an hour no reaction with iodine is obtained. 

 At this stage the drops from tube B still give a clear indication of the presence 

 of starch. A typical result is given in Table XXI. on p. 125. 



A suitable exercise is now to estimate the amount of reducing sugar present 

 in each tube. 



47. Estimation of the Relative Activity of an Enzyme (Rennin). 

 Material required, (i.) Boiled milk, to which has been added one-tenth of 



its volume of 10 per cent, calcium chloride solution, (ii.) Arbitrary standard 

 of activity — prepared by diluting either commercial " Essence of Rennet "" or 

 Benger's "' Liquor Pepticus " to such a strength as will produce the curdling 

 of an equal volume of CaClo milk mixture in about 10-12 minutes at room 



