PROSTHETIC GROUPS, COENZYMES AND ENZYMES 



recently been made over the pH range 5.3 to 10, and in addition 

 the influence of some electrolytes was studied (28). Again 

 fluorescence measurements were found to be superior to spec- 

 trophotometry. DPNH fluoresces but DPN does not. The 

 sensitivity of the method is so great that a reaction cycle in- 

 volving a concentration change of DPNH less than Iju molar 

 can easily be recorded. 



It was found possible to calculate all six of the constants 

 from equations (4) and {4a) from initial reaction velocities in a 

 test system with systematic variations in the concentrations of 

 DPN, DPNH, alcohol, and aldehyde. Some data are sum- 

 marized in Table HI. 



Let us first consider the influence of varied pH. The "on" 

 velocities for DPNH (ki) and DPN (k^) both show a maximum 

 at pH 7 to 8, and decrease toward alkaline and acidic reaction. 

 The decrease on the alkaline side follows monovalent dissociation 

 curves with pK' around 9 for both ki and k^. This drop is 

 reasonably explained, in analogy with O.Y.E., by assuming 

 that the pyrophosphate group of DPN(H) is linked to positively 

 charged groups, perhaps again — NH3, in the protein. Kaplan 

 and Ciotti (5) have recently shown that splitting the pyro- 

 phosphate group by snake venom pyrophosphatase releases 

 reduced nicotinamide mononucleotide from DPNH-ADH, 

 and they concluded that the pyrophosphate grouping is essential 

 for the attachment of DPNH to the liver enzyme. 



Toward the acidic side k^ drops sooner (/'K' = 6.5) than 

 ki (pK' = 5.5). The reason for this we do not know, nor do 

 we understand entirely why ki is 10-100 times higher than k^. 

 We think both facts are connected with the extra plus charge 

 in the pyridinium ring of DPN ^ and with the presence of two 

 more or less positively charged atoms of zinc in the ADH 

 molecule. 



The "off' velocity of DPNH (A;2) is nearly independent of 

 />H from 7 to 10, but this is by no means true for the DPNADH' 

 (kz). The ks values lie very close to a monovalent dissociation 

 curve with pK' = 7.8. Both k2 and kz decrease below pH 7 



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