244 CUVETTE COLORIMETRY 



and place in a desiccator over calcium oxide in the dark. Make 



up the soln. just before use. 

 1.25% Potassium Ferricyanide. Prepare just before use. 

 Standard Solutio)i. Prepare fresh each week a soln. of 1 mg. allan- 



toin/ml. 



PROCEDURE 



1. Into a Pyrex test tube (125 X 9 mm. inside) provided with a 

 ground-glass stopper place 10 mg. dry enzyme powder, 0.5 ml. 

 carbonate-cyanide soln., 2 ml. of the sample to be analyzed, and a 

 drop of chloroform. 



2. Stopper the tube and let stand at 37° for 12 hr. An occasional 

 shaking aids in the solution of the enzyme. 



3. Transfer 2 ml. of the mixture into another test tube and add 

 0.2 ml. 10% trichloroacetic acid and 0.1 ml. 2% sodium tungstate. 

 Mix after stoppering by inverting a few times. Then add 0.1 ml. 

 iV/15 sulfuric acid and again mix by inversion. 



4. Place tube in a large beaker of water at room temperature 

 and heat the water quickly to 90° for 5 min. Cool quickly in ice 

 water for 2 min. 



5. Add 0.3 ml. 0.5% phenylhydrazine soln., stopper and shake 

 vigorously. 



6. Set in a water bath at 60° for 5 min. and quickly cool in ice 

 water. 



7. Centrifuge; carefully float a drop of alcohol on the surface 

 of the liquid, and again centrifuge to throw down floating particles. 



8. Carefully pipette 2 ml. clear supernatant into another dry test 

 tube and cool it in a dry ice-alcohol mixture contained in a beaker 

 surrounded by an ice-salt bath maintained at — 15 to — 20°. 



9. Cool some cone, hydrochloric acid in the Dry Ice-alcohol 

 mixture, and, after the tube has been cooling for about 10 min., add 

 1.5 ml. of the cold hydrochloric acid to it. 



10. Stopper, and continually invert in the air until the frozen 

 material melts. 



11. Just before the last of the frozen material disappears add 0.2 

 ml. potassium ferricyanide soln. and mix quickly by several inver- 

 sions. 



12. Set in an ice-salt bath for 5 min. and then place the tube in 

 a beaker of water at room temperature. 



