Discussion 



87 



Another criterion for the oxidation state of pyridine nucleotide in 

 mitochondria which we have recently applied in some detail (Chance, B., 

 and Baltscheffsky, H. (1958). J. biol. Chem., 733, 736) is that the oxi- 

 dized form of DPN has negligible fluorescence and the reduced form has 

 intense fluorescence, with an emission maximum at 470 m^x for the free 

 form and at 443 my. for the bound form (Boyer, P. D., and Theorell, H. 

 (1956). Acta chem. scand., 10, 447; Duysens, L. N. M., and Amesz, J. 

 (1957). Biochim. biophys. Acta, 24, 19). We have recently investigated 

 the fluorescence of mitochondria in the absence of ADP (state 4), and 

 find a maximum at 443 mji. which diminishes upon addition of ADP 

 (Fig. IB). 



In sunmaary, spectrophotometric and fluorometric data indicate the 



active -resting 



' I ' I ' I ' I ' I ^ 



400 420 440 460 480500 580 600 



Mmp) 



Fig. IB (Chance). (Expt. no. 777) 



presence of a form of reduced pyridine nucleotide in the mitochondria, 

 the concentration of which is diminished by ADP and increased by 

 amytal. This form, therefore, appears to be identical with the com- 

 pound which Prof. Slater is attempting to assay enzymically. Unless he 

 can present physical data proving that he is studying a form of oxidized 

 DPN which has the fluorescence and absorption properties of reduced 

 DPN, it seems appropriate that he consider the possibility that he is 

 studying the DPNH^I compound rather than the DPN^I compound 

 as an explanation for his experiment. 



[Note added in proof. For some time there has been evidence that the DPNH -^ I 

 compound of mitochondria may not be as active towards certain dehydrogen- 

 ases as is free DPNH: "Upon addition of acetoacetate to the mitochondria in 

 the resting state, 4, the extent of oxidation of DPNH is very small compared 

 with that which is obtained upon addition of acetoacetate to state 3." (Chance, 

 B., and WilHams, G. R. (1956). Advanc. EnzymoL, 17, 107).J 



