298 Freed, Reitlicl, and Rcjninert 



peroxidase found in this laboratory. It would appear rather that there 

 may be one of two alternative consequences of this adsorption: (a) the 

 adsorption restricts energy transfer to the protein molecule, thus modi- 

 fying the rate of reaction, or (b) the adsorption of the solute species 

 results in a modification of the structure of the protein such that the 

 kinetics of the reaction it catalyzes is changed (29, 32). 



It has been suggested that inasmuch as many proteins contain flu- 

 orescent centers, the fluorescence intensity of such a species might cor- 

 relate with enzymatic activity. Indeed such has been shown to be the 

 case (21, 31). It has been suggested by Karreman et al. (16) that the 

 fluorescent emission is the mechanism by which energy may be trans- 

 ferred from an enzyme to its substrate molecule. It would appear, 

 therefore, that if 2,4-D reduced the fluorescence intensity of the en- 

 zyme, this would indicate interference with energy transfer. Also, if 

 the enzymatic activity of the protein were affected by the concentra- 

 tion of 2,4-D, which reduces fluorescence intensity, this would indi- 

 cate that adsorption interferes with energy transfer. Upon testing this 

 theory with glyceraldehyde phosphate dehydrogenase and a-amylase, 

 no discernible effect on enzymatic activity was found at a concentra- 

 tion of 2,4-D at which fluorescence intensity was markedly reduced. 

 This measurement was made using the Aminco-Bowman spectrophoto- 

 fluorometer. 



Inasmuch as reduction in fluorescence intensity did not correlate 

 with the change in enzymatic activity, it was felt that the effect of the 

 chemical in modifying the enzymatic activity of the protein Avas not 

 due to restriction or modification of energy transfer within the pro- 

 tein. From these considerations the assumption was made that the 

 chemical exerts its influence by modification of the structure of the 

 enzyme molecule. It is a well-known phenomenon that the modifica- 

 tion of a catalyst's surface materially changes the property of the sm- 

 face as a catalyst. In order to study this problem, attention was then 

 turned to finding an enzyme, the activity of which was known to be a 

 function of the structinal integrity of the protein molecule. Peroxi- 

 dase (horse-radish) has been cited as an example of a protein capable 

 of undergoing reversible denaturation with heat. Thus, the degree of 

 reversible denaturation of peroxidase may be followed by measure- 

 ment of its enzymatic activity as a function of temperature. It was 

 reasoned that if the adsorbed 2,4-D brought about a change in the 

 structure of the molecule, this should facilitate the heat denaturation 

 of peroxidase. Accordingly a study of the rate of change of horse- 

 radish peroxidase activity with increasing temj>crature witli and with- 

 out 2,4-D was followed. The results of this study are shown in Figure 2. 



