DIPHQSPHOPYRIDINE NUCLEOTIDE 



397 



Fig. 136. Apparatus used in extraction of DPN from frozen dried sections: 

 A, micro centrifuge tube with frozen dried section; B, tube after addition of 

 water; C, tube sealed off during heat inactivation ; D, tube during extraction 

 of DPN. From Anfinsen (WW 



4. Plunge sealed tube into boiling water, and, after 2 min., re- 

 move and chill well in ice water. 



5. Centrifuge down drops of soln. left on the sides and top. The 

 special centrifuge cup (£") is convenient for this purpose. Open tube 

 with a file. 



SPECIAL REAGENTS 



O.lSIf M Sodium Bicarbonate saturated with a mixture of 5% carbon 

 dioxide and 95% nitrogen. 



0.003 M Disodium Hydrogen Arsenate. 



0.016 M (approx.) Sodium Hexose Diphosphate (o 1 mg. phospho- 

 rus/ml.). Prepare from the calcium salt by adding about 700 mg. 

 of the latter to 40 ml. of 1 % oxalic acid. After shaking neutralize 

 the mixture with sodium bicarbonate to chlorophenol red. De- 

 colorize with charcoal and filter. Test filtrate for oxalate, and, if 

 present, precipitate it with a little solid calcium hexose diphos- 

 phate and refilter through the original filter paper. Determine the 

 phosphorus content of the filtrate and dilute the soln. until it con- 

 tains 1 mg. organic phosphorus/ml. The presence of 3-4% in- 

 organic phosphorus may be ignored. Store in refrigerator where 

 the soln. w^ill be stable for several months. 



