44 A SYMPOSIUM ON RESPIRATORY ENZYMES 



enzyme. By selecting proper concentrations any reaction in this 

 scheme can be made the hmiting factor. Thus by measuring the rate 

 with which cytochrome c is reduced, it is possible to determine rates 

 of reaction and to demonstrate formation of enzyme complexes for 

 any of the reactions involved. Negelein and Haas have shown by 

 ultraviolet spectroscopy the formation of a complex between protein 

 I, triphosphopyridine nucleotide, and glucose-6-phosphate. With the 

 method indicated above the dissociation of the cytochrome reductase 

 into alloxazine mononucleotide and protein II can be demonstrated 

 if to a small but constant amount of protein II increasing amounts of 

 alloxazine mononucleotide are added. 



The dissociation constant of cytochrome reductase is small 

 (K = 1 X 10"^ M), and under physiological conditions this enzyme 

 will therefore be present as the practically undissociated complex. 

 The value of the spectrophotometric method for the study of enzyme 

 reactions is well demonstrated here, since 10"'' mg. of flavin are 

 suflBcient for accurate determinations. Not only the relation between 

 the prosthetic group and protein of one enzyme may be studied 

 with this system, but also the interaction of two different enzymes. 

 For example, we can measure the rate of reaction when increasing 

 amounts of dihydrotriphosphopyridine nucleotide are added to a 

 constant amount of cytochrome reductase. The velocity of the reduc- 

 tion of the enzyme is given by 



(1) d(CR)_ kr(T) (CR) 



dt ~ {T)+Kd 



in which ( T) and ( CR) are the concentrations of triphosphopyridine 

 nucleotide and cytochrome reductase, respectively, K is the first- 

 order velocity constant, and Kd the dissociation constant of the 

 pyridine-alloxazine complex. Rate constants and dissociation con- 

 stants have been determined at different temperatures, and by apply- 

 ing the Arrhenius equation the energy of activation is found to be 

 about 10 kg. cal., and the heat of dissociation about 2 kg. cal., for 

 the reaction in which cytochrome reductase is reduced by dihydro- 

 triphosphopyridine nucleotide. 



Similar experiments have been made for the purpose of studying 

 the oxidation of cytochrome reductase by cytochrome, and again 

 the formation of a complex between the two reaction partners could 

 be established. Furthermore, from the results of the kinetic deter- 

 minations it can be concluded that in the course of the oxidation of 

 alloxazine free radicals are involved. This could almost be antici- 



