638 Discussion 



In our newer mechanism, we write I in a reduced form IH.2, so that DPN ~ I 

 becomes DPN ^^ IH, which is meant to represent a compound of oxidized DPN with 

 IH2. As before, we envisage that it is formed during the oxidation of DPNH to DPN+. 



The only difference between the new formulation and the old is that the 'I' attached 

 to the DPN+ is now written with an oxidizable H atom. We regard it as a compound 

 between DPN and IHj, rather than between DPNH and I, since we write its cleavage as 



DPN ~ IH + H+ ^ DPN+ + IHo (iv) 



If by DPNH ~' I, Chance means DPNH in which one of the two hydrogen atoms 

 on the 4-carbon atom of the pyridine ring is replaced by I, there is no difference 

 between his views and ours. But we should prefer to regard this as a DPN compound 

 rather than a DPNH compound. 

 Chance: Slater's Equations (i) and (ii) require a few comments, in view of the observa- 

 tions of Purvis that a-oxoglutarate, glutamate or succinate increases the concentra- 

 tion of the hypothetical DPN ~ I {Nature, Loud. 182, 711, 1958). Equation (i) 

 suggests that DPN ~ I is formed in an oxidation reaction, but this appears to 

 be a serious inconsistency. The material which is observed to increase in absorption 

 upon addition of glutamate or succinate to mitochondria absorbs maximally at 

 340 m/{ and fluoresces maximally at 443 nyi (Chance, in Proc. Ciba Foundation Sym- 

 posium on the Regulation of Cell Metabolism, 1959, p. 86). However, addition com- 

 pounds similar to cyanide (Equation (ii)) absorb maximally at shorter wavelengths 

 (cf. Van Eys, Stolzenbach, Sherwood, and Kaplan, Biochim. biophys. Acta 27, 63, 

 1958). Thus, Equation (ii) appears to be inconsistent with the direct absorption data. 

 Addition compounds with ketones contain a reduced state of DPN as they resemble 

 charge transfer complexes (Burton, San Pietro and Kaplan, Arch. Biochem. Biophys. 

 70, 87, 1957). 



We have continuously recognized that Equation (iii) is unlikely, have never published 

 such an equation, and have stated since 1955 (Chance, Williams, Holmes and Higgins, 

 J. biol. Chem. 217, 443, 1955): "It should be noted that the energy for the formation 

 of the '— ' I need not be conserved in the oxidation-reduction reaction of Equations 8 or 

 10; it could have been conserved by the enzyme in a previous reaction cycle, in the 

 chemical structure designated by the asterisk.' (See also Slater, this volume, p. 622.) 



I would accept a charge transfer complex as a possible form of DPNH -^ I in 

 which the reducing equivalents resonate between the tv.'o structures: 



DPNH ~ I v^ DPN ~ IH 



provided the resonance favoured the former configuration sufficiently to agree with 

 the physical data on absorption and fluorescence. This is, however, equivalent to 

 saying that the physical data at present require a configuration not measurably different 

 from DPNH — I. It is clear that isolation of such a compound is desirable. 

 Slater: I do not consider that there is any principal difference between the structure of the 

 DPN-cyanide compound, and that of DPN-ketone compounds. The formation of 

 both compounds may be described by the equations 



H H+ H. .A 



cr^y^o 



r I I 



where A" is CN^ or CHs.CO.CHg^ (for acetone), respectively. DPN-A can be 

 regarded as an addition compound of DPN+ and A", which I should prefer, or as 

 a DPNH compound in which the H is replaced by A. It cannot be regarded as an 

 addition compound of DPNH with A. All compounds of the type DPN-A have an 

 absorption band in the near ultra-violet region. This is at 340 nyi when A is H — or 

 CH3.CO.CH2— , at 330 m/< when A is RS— , and at 325 m/i when A is CN-. Thus 

 DPNH and the DPN-acetone compound cannot be distinguished spectrophoto- 

 metrically and in fact Warburg mistakenly identified the similar DPN-glyceraldehyde 



