140 A SYMPOSIUM ON RESPIRATORY ENZYMES 



thetic group and the total concentration (combined and uncombined) 

 may be regarded as the same. If this assumption cannot be made, 

 then a somewhat more comphcated equation must be used (see ref. 

 19). 



The spHtting of the flavoprotein has been accomphshed in two 

 ways. Theorell (26) separated the riboflavin phosphate and the 

 protein by a 72-hour dialysis against a dilute acid solution. By 

 adding the riboflavin phosphate to the protein remaining after the 

 dialysis, the original activity was restored. Warburg and Christian 

 (27) later developed a simpler and more rapid method in which 

 the solution containing the flavoprotein is acidified in the presence 

 of large concentrations of ammonium sulfate. In this solution the 

 flavoprotein is split and the protein is precipitated. The ammonium 

 sulfate protects the protein moiety against denaturation. 



The known dissociation constants, determined in the manner 

 previously outlined, are presented in Table 2. 



Table 2.— Dissociation constants 



Enzyme Dissociation constant Reference 



Amino acid oxidase 250X10-9 28 



Old yellow enzyme 60X10-» 2 



New yellow enzyme 27X10-9 12 



Cytochrome c reductase 1 X 10"' 19 



In comparing the activities of various enzymes, several factors 

 must be taken into account. These are (1) the concentrations of the 

 reacting substances, (2) the aflBnity of the enzyme for the reacting 

 substrate molecules, and (3) the absolute reaction velocity of the 

 reactants when in the form of the protein-substrate complex. The 

 last two of these factors determine the intrinsic activity of the 

 enzyme, but in determining how great a role any enzyme plays in a 

 given cell, the concentrations are of prime importance. 



All the evidence indicates that in every case substrate and enzyme 

 form a complex before reaction sets in. As an example we may 

 consider the reaction between reduced triphosphopyridine nucleo- 

 tide (TPNH2) and oxidized cytochrome c reductase (CR). 



( 2 ) TPNH, -f CR = TPN -f CRH2 



A complex is first formed and the velocity of the reaction is pro- 

 portional to the concentration of this complex. 



