IV. BIOCHEMICAL SYSTEMS 511 



of fructose diphosphate and the triose phosphates either singly or together, 

 depending on the order of addition of the three enzymes concerned. This 

 may be considered an example of the way in which substances other than 

 DPX may be quantitated by linking the reactions to DPN-dependent 

 enzyme systems. 



DPN may also be converted chemically to DPNH2, using hydrosulfite 

 reduction (p. 491), and measured by absorption at 340 m/i.'*^ This procedure 

 does not differentiate between DPN and TPN. Cyanide also will react 

 with DPN (p. 487) to form a complex with an absorption maximum at 

 340 m^ This reaction may be used analytically for DPN, as shown by 

 Colowick et al.^^^ 



DPNH2 may be quantitated by oxidation with pyruvic acid and lactic 

 dehj^drogenase, the disappearance of absorption at 340 mfj, being a measure 

 of the amount of reduced coenzyme present. ^^^ 



3. TPN 



TPN can be determined spectrophotometrically in an isocitric dehydro- 

 genase system which specifically requires TPN.*° This system, as described 

 by Ochoa,^^ is dependent on two reactions, the net result of which is: 



Mn++ 



D-Isocitric acid + TPN ^— a-Ketoglutaric acid + CO. + TPNH. 



Oxalosuccinic acid is an intermediate in the reaction. 



TPN may also be determined utilizing the glucose dehydrogenase reac- 

 tion originally described by Warburg and Christian,^ . 7 , i84 , i85 ^y-hich jg also 

 TPN-dependent. 



Glucose-6-phosphate + TPN ;^ 6-Phosphogluconic acid + TPNH2 



This reaction can be coupled to a system in which cytochrome c is the 

 final hydrogen acceptor to form a very sensitive assay procedure.^^^ It 

 may also be linked to the system 



hexokinase 



Glucose + ATP ^ ^=^ Glucose-6-phosphate + ADP 



to supply glucose-6-phosphate for the TPN-dependent reaction listed above. 

 If ATP is the limiting factor in the mixture, then the amount of TPN re- 

 duced in the combined reaction will have a proportionality to the amount 

 of ATP present, permitting a quantitative ATP assay. "^ 



18' G. A. LePage, J. Biol. Chem. 168, 623 (1947). 



i»2 S. P. Colowick, N. O. Kaplan, and M. M. Ciotti, /. Biol. Chem. 191, 447 (1951). 



»" F. Kubowitz and P. Ott, Biochem. Z. 314, 94 (1943). 



'8* O. Warburg and W. Christian, Biochem. Z. 242, 206 (1931). 



186 E. Negelein and W. Gerischer, Biochem. Z. 284, 289 (1936). 



'86 E. Haas, C. J. Harper, and T. II. Hogness, J. Biol. Chem. 142, 835 (1942). 



