398 GASOMETRIC-MANOMKTRIC METHODS 



Muscle Extract. Remove the fascia as well as possible from muscles 

 of the hind legs and back of a cat. Homogenize thoroughly with an 

 equal weight of water and crushed ice, and centrifuge. A Waring 

 blendor and a Sharpies centrifuge are useful for these steps. With 

 stirring, add 4 vol. ice-cold acetone to the soln. in a slow stream. 

 Let the precipitate stand for 30 min. in the cold ; decant the super- 

 natant, and centrifuge the remainder. Wash the residue twice with 

 cold acetone in the centrifuge, transfer to a Biichner funnel anrl 

 wash with acetone and ether. Break up the cake into small pieces 

 and dry overnight in vacuo over sulfuric acid. Prepare a paste 

 of this acetone powder by grinding 600 mg. with successive 2 ml. 

 portions of water until 20 ml. has been added. Centrifuge; dialyze 

 the supernatant liquid in the cold against running distilled water 

 for 24 hr., and centrifuge. Remove interfering nucleotides by 

 adding 600 mg. of Norit charcoal to the dialyzed soln. Shake 

 mechanically in the cold for 1 hr., centrifuge, and treat the super- 

 natant fluid with another portion of charcoal for 1.5 hr. Centri- 

 fuge and filter. The DPN blank for this light brown soln. is small 

 at first and disappears completely after 12-24 hr. at 6°. The 

 acetone powder is very stable when kept in vacuo in the cold. The 

 activity of the aqueous extract remains constant 4-5 days when 

 kept cold. 



PROCEDURE 



1. Combine 0.4 ml. bicarbonate soln. with 0.6 ml. hexose diphos- 

 phate and 0.3 ml. arsenate. To 1 vol. of this mixture add 0.17 vol. 

 DPN soln., either the standard or unknown soln. 



2. Pipette 1.60 //.l. resulting soln. into the diver. 



3. Make up to a total vol. of 3.14 fx\. in the diver with water and 

 muscle extract (1.0 ml. muscle extract equivalent to 30 mg. acetone 

 powder). About 1 vol. muscle extract, diluted with 0.5 vol. distilled 

 water will probably be required. 



4. Flush the diver with the gas mixture containing 5% carbon 

 dioxide and 95% nitrogen for 3 min. to bring the pH to 7.4. 



5. Seal the neck of the diver with paraffin oil saturated with the 

 gas mixture and transfer to the flotation tube. 



