296 



Freed, Reithel, and Remmert 



Table 5. Washing as a factor in the reversal of 2,4-D inhibi- 

 tion of oxygen uptake by mitochondria. 



two 2,4-D was added to assure that the activity was still 2,4-D sensi- 

 tive. The results of this study are to be found in Table 5. 



It will be noted that exposure of the mitochondria to 2,4-D fol- 

 lowed by washing to remove the chemical, resulted in a complete re- 

 covery of the activity as measured by oxygen uptake. However, addi- 

 tion of 2,4-D to the Warburg vessel demonstrated that the mechanism 

 responsible for oxygen uptake was still 2,4-D sensitive. A similar situa- 

 tion was to be found with the washed control. It should be noted in 

 passing, however, that long exposure of the mitochondria to 2,4-D, 40 

 min. or more, resulted in an irreversible loss of their oxidative 

 capacity. 



The behavior noted with these mitochondria strongly supports 

 the theory that the primary event in the action of these chemicals is 

 adsorption. It is obvious that had this chemical reacted with con- 

 stituents of the mitochondria to form a chemical complex or com- 

 pound, simple washing would not have removed a sufficient amount 

 of the chemical to restore the full oxidative capacity of the mitochon- 

 dria. On the other hand, if this chemical were simply adsorbed by 

 physical forces on the surface of the enzyme, one would expect that 

 washing would remove substantially all of the 2,4-D, thus permitting 

 recovery of the oxidative ability. This apparent adsorption on the 

 surface of protein also affords a possible explanation of why 2,4-D 

 gives a rather general nonspecific inhibition of oxygen uptake of 

 mitochondria using different members of the Krebs cycle intermedi- 

 ates. In seeking further evidence in support of the theory that 2,4-D 

 is adsorbed by proteins, a study of 2,4-D adsorption by proteins was 

 made by equilibrium dialysis. This study clearly demonstrated the 

 adsorption involving four molecules of chemical per molecide of 

 protein. 



One of the characteristics of an adsorbing system is tiiat less of 

 the solute species will be adsorbed by a surface as temperature in- 

 creases (9). Therefore, it should be possible to ascertain whether or 



