PROSTHETIC GROUPS, COENZYMES AND ENZYMES 



fluorescence measurements it is possible to work in much more 

 dilute solutions, in the case of FMN down to 10~^ M. As a 

 matter of fact the region lO"*^ to 10~^ M is even optimal for 

 quantitative fluorescence work, since at concentrations higher 

 than 10~^ M certain deviations caused by light absorption in the 

 cuvette become appreciable. 



The possibility of working in very dilute solutions and 

 varying the concentrations by a factor of 10^ simply by changing 

 the sensitivity of the amplifying system was found to be sur- 

 prisingly valuable. The coinbination reaction ("on reaction") 

 between FMN and apoprotein is very rapid, under certain con- 

 ditions of the saine order of magnitude as for DPNH + ADH. 

 But at the high dilutions that we can now apply, the reaction 

 velocity could be accurately studied without the aid of "rapid- 

 flow" apparatus and oscillograph. 



From the siinple relationship 



1 FMN + 1 apoprotein , ' ^ 1 O.Y.E. 



one might expect A"i to be of second order, /:.> of first order. 

 However, this could not be taken for granted since more than 

 one group in FMN combines with the protein. Thus it was con- 

 ceivable that half-coupled intermediates could be formed and 

 disturb the simple kinetic relations. However, this was found 

 not to be the case. It was concluded that if half-coupled 

 intermediates existed, they reacted far too rapidly to interfere 

 with the kinetics of the over-all process. 



THE DISSOCIATION VELOCITY (27) 



First of all, to our surprise we found that the dissociation 

 velocity and the degree of dissociation of O.Y.E. in pure water 

 or in solutions with anions which forin weak acids was im- 

 measurably low. Increasing the acidity down to /;H 4 in ace- 

 tate (at still lower pH the O.Y.E. begins to get destroyed), 

 or increasing the pH to 1 1 in glycine, still caused no dissociation 

 of O.Y.E. However, when salts of strong acids were added, the 

 enzyme was found to dissociate readily. 



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