PYRIDINE NUCLEOTIDES IN MITOCHONDRIA 21 5 



membrane was not then accepted by these workers. Because the intra- 

 mitochondrial DPNH could not be oxidized in the inhibited State 4, 

 Chance and WiUiams [7] and Chance and H. Bahscheffsky [18] concluded 

 that DPNH was in an inhibited form DPNH ~ I. Chance and Hollunger 

 [19] have recently stated, "It is further concluded that the reduced form 

 is bound to some ligand, for example, a protein, because the fluorescence 

 maximum is at 443 m/x. Thus this material is denoted DPNH ~ I. . . . "* 



It is doubtful whether the sort of chemical bonds which Huennekens 

 and Green [2] and Chance and Hollunger [19] have in mind could sur- 

 vive the treatment with acid or alkali used to prepare deproteinized 

 extracts for determinations of the pyridine nucleotides. It is probable, 

 therefore, that these bound forms would yield free DPN + and DPNH in 

 the extracts. We were more concerned with the possibility that a stable 

 compound of DPN of low molecular weight, which could survive either 

 the acid or alkali extraction, might be present in mitochondria. 



Purvis [10, 11] found that the total amount of DPN and TPN, deter- 

 mined by incubation of rat-liver mitochondria with P;, ADP + Pi, or 

 dinitrophenol, appreciably exceeded the amounts of (DPN '+ DPNH) 

 and of (TPN+ + TPNH), respectively, determined in the fresh mito- 

 chondria (see Table I). The amount of "extra DPN" found in this way 

 averaged i • 10 /imoles g. protein for the Amsterdam preparations and 

 1-37 yumoles/g. protein for the Brandeis preparations. The corresponding 

 values for "extra TPN" were 2-50 and i -42, respectively. Only two out 

 of sixty-three preparations examined did not show any of this material. 



Klingenberg and Slenczka [8], who did not find any evidence for 

 "extra DPN", in somewhat different experiments, concluded that Purvis' 

 results were due to a failure of his fluorimetric procedure. 



Table I shows that we also, on the average, find little if any excess of 



* The difference spectrum (anaerobic minus aerobic steady state) shows a 

 pyridine nucleotide peak at 320 m/:x rather than at 340 m/i [16 (see Fig. i), 15, 17]. 

 This does not, however, prove that the DPNH found in the mitochondria is 

 "bound " in such a way as to cause a displacement of the absorption peak, as un- 

 fortunately appears to be implied by Holton et al. [15]. Biicher and Klingenberg 

 [20] have with justification criticized this conclusion, which in fact we had not 

 intended should be made from our results, since the displacement of the 340-m;n 

 peak might be caused by the contribution of the S-bands of the cytochromes in 

 this region of the spectrum, as already discussed by Holton [12]. Chance and M. 

 Baltscheffsky [17], Chance [21], and Chance and Hollunger [19] have pointed out 

 that the DPNH which appears when ADP is exhausted by a respiring mito- 

 chondrial preparation has an absorption peak at 340 m/x. The changes in the degree 

 of reduction of the cytochromes is much less than that of the DPN under these 

 conditions, so that the contribution of the cytochrome S-bands to the spectrum 

 in the near ultraviolet would be much less. It should be pointed out, however, that 

 Chance and M. Baltscheffsky [17] are of the opinion that the displacement of the 

 DPNH peak in the anaerobic spectrum cannot be explained by the S-bands of the 

 cytochromes. 



