39b GASOMETRIC-MANOMETRIC METHODS 



7. For the determination of both components in a mixture, first 

 measm'e the cocarboxylase. Then determine the thiamine in a sepa- 

 rate experiment by adding, to the 0.2 /xl. imknown, 0.2 //,!. cocar- 

 boxylase soln. containing sufficient of the substance to make the 

 total in the diver 0.001 /xg. 



DIPHOSPHOPYRIDINE NUCLEOTIDE 



Anfinsen (1944) adapted the method of Jandorf, Klemperer, and 

 Hastings (1941) to the measurement of diphosphopyridine nucleo- 

 tide ( DPN) in microtome sections of tissue by the use of the Carte- 

 sian-diver technique. The method of Anfinsen permits the estima- 

 tion of 1-6 m/xg. DPN with an error of less than 5% ; this represents 

 a thousandfold increase in the sensitivity of the Warburg procedure 

 of Jandorf et al. The principle of the method lies in the fact that the 

 enzymatic conversion of hexose diphosphate to phosphoglyceric acid 

 and phosphoglycerol can be made to take place under conditions 

 in which the quantity of DPN present is the limiting factor. These 

 conditions require the presence of a muscle extract to supply en- 

 zymes and arsenate to limit specifically the glycolytic process. The 

 dearsenylation of the l-arseno-3-phosphoglyceric acid formed occurs 

 spantaneously. The molecule of acid produced in this step is made 

 manifest by the liberation of carbon dioxide from a bicarbonate 

 buffer; thus the gas evolution becomes a measure of the concentra- 

 tion of the DPN. 



Anfinsen Method for Diphosphopyridine Nucleotide 



Extraction of DPN from Tissue Sections. Since DPN is 

 rapidly destroyed by concomitant enzymes in the tissue, the follow- 

 ing procedure is employed for the extraction: 



1. Weigh frozen-dried sections on a quartz torsion balance (page 

 191) and transfer to micro centrifuge tubes (Fig. 136 A), made of 3 

 or 4 mm. tubing. 



2. Draw out tube to form a constriction {B). 



3. Add a known amount of distilled water with a constriction 

 pipette (page 172) and rapidly seal the tube (C). 



