IV. BIOCHEMICAL SYSTEMS 509 



linked enzyme systems or on the respiration of rat liver in the presence of 

 fumarate or pyruvate. 



M. ASSAY PROCEDURES FOR DPN AND TPN 

 Chemical methods have been described elsewhere (p. 537). 



1. General Principles 



A number of enzymatic methods have been described which permit a 

 quantitation of DPN or TPN. The most widely used are the spectrophoto- 

 metric techniques which depend upon the fact that oxidized DPN-TPN 

 exhibit no absorption at 340 m^ whereas the reduced enzymes absorb 

 strongly and identically at this wavelength. Since the molecular extinction 

 coefficients are known (p. 490) and since absorption is proportional to con- 

 centration, these coenzymes can be readily quantitated. The methods are 

 fairly sensitive. There are few substances, either components or reaction 

 products of the enzyme systems, which have interfering light absorption 

 at 340 mju. Determinations may be made quickly with small quantities of 

 materials and are especially useful in following the progress of reactions. 

 The methods may be adapted to measure either the appearance or disap- 

 pearance of the reduced forms, depending on whether the particular enzyme 

 system donates or accepts hydrogen. They may also be used to measure 

 the concentration of the apoenzymes or the substrates when these are 

 limiting factors in the reaction mixtures. Specificity for DPN or TPN can 

 be attained when dealing with mixtures by using an apoenzyme and a 

 substrate which specifically requires one or the other coenzyme. These 

 systems also permit the measurement of reactions not amenable to direct 

 spectrophotometric determination if they can be coupled to reactions de- 

 pendent on these coenzymes. ^""^'^^ 



2. DPN 



As an example, DPN may be determined as described^y Romberg,"^ 

 using crystalline alcohol dehydrogenase and alcohol to reduce the DPN. 

 The components of the test system are ethanol 0.3 ml., glycine (1.5%) 0.2 

 ml., sodium pyrophosphate buffer (0.03 AI, pH 8.5) 1.5 ml., alcohol de- 

 hydrogenase 5 to 10 7, and water or unknown q.s. 3.0 ml. Figure 8 shows 

 the sensitivity and proportionality of the method with known amounts 

 of DPN. The reaction was complete in 5 minutes. The specificity of the 

 procedure could be verified by observing the complete disappearance of 



>" O. W:irl)urg, Ergeb. Enzymforsch. 7, 210 (1938). 



•'^ E. Schleiik, in Methoden der Fermentforscliung. Academic Press, New York, 1945. 



'79 T. R. Hogness and V. R. Potter, Ann. Rev. Biochem. 10, 509 (1941). 



