BRITTON CHANCE 



cell or mitochondrial suspensions is so great that this high per- 

 formance is adversely affected and as much as 10"^ mole of 

 reactants must be expended to measure rapid reactions. At the 

 same time we have discovered a method for increasing the fluid 

 economy by 100-fold without sacrificing the performance, and 

 thus it is now possible to study the rapid reactions of cytochromes, 

 flavoproteins, and pyridine nucleotides in relatively small 

 quantities of mitochondria or intact cells. 



The new method is called the regenerative-flow method 

 (21,24). It depends upon the fact that the reduced state of a 

 respiratory system is regenerated shortly after a low concentra- 

 tion of oxygen has been added, and the process may be repeated 

 in a completely reversible fashion. Thus an enzyme solution 

 that has once been reacted with oxygen can soon be used again 

 for a second reaction with oxygen. A given volume of mito- 

 chondrial suspension will suffice for a large number of kinetic 

 experiments if (7) the apparatus is designed for re-use of the 

 regenerated material and (2) the material is diluted only slightly 

 by the addition of oxygen solution. 



A practical design of an apparatus for this purpose is il- 

 lustrated in Figure 6. An 80-ml. syringe containing the anaero- 

 bic reduced mitochondrial suspension is discharged at the same 

 time as a 1-ml. syringe containing the solution of oxygen. These 

 reactants enter a mixing chamber (6 jets) and thence into a 1 

 X 1 X 1 cm. observation tube by means of a pushing block. 

 The effluent is received in an 80-ml. syringe in which the oxi- 

 dized components are allowed to become reduced again by 

 their own respiration. The solution in the bottom syringe is 

 then discharged into the upper one, the small syringe being 

 refilled with oxygen solution. The measurement of reaction 

 kinetics can be repeated at wavelengths appropriate to another 

 component of the reaction sequence. This process can be re- 

 peated until the dilution of the solution becomes objectionable — 

 usually 40 experiments are possible with one filling of this 

 apparatus. 



328 



