BRITTON CHANCE 



41 



of the enzyme substrate compound. Both these quantities can be measured 

 accurately and for smaller ratios of ao and Xo and low enzyme concentra- 

 tions ; the half-life of the enzyme substrate compound is long enough to be 

 measured by very simple methods; no flow apparatus being required. 



Fig. 2. Gibson and Rough- 

 ton's thermal data on reactions 

 of hemoglobin with various lig- 

 ands (7). In the terminology of 

 Gibson and Roughton, the 

 primed values refer to 'on' ve- 

 locity constants, the unprimed 

 to 'off' velocity constants. The 

 subscripts refer to the binding 

 of the first, second, third and 

 fourth molecule of the particu- 

 lar ligand and the letters k, 1, 

 and n refer respectively to O2 , 

 CO, and NO molecules. Data 

 from Faraday Society. 



O 



20.000 

 Enerqy of activotion (cal) 



The equation, which has been derived elsewhere (16), is as follows: 



ki = 



1 



(e — Pm) tioff 



(-5) 



where e is equal to the initial concentration of the enzyme, p,n is maximum 

 concentration of the intermediate compound under particular experi- 

 mental conditions (this is also the steady state concentration) and t^off 

 is the time interval from the formation of the enzyme substrate compound 

 until its concentration has fallen to half its maximum value. Figure 3 

 serves to illustrate the measurement of these quantities. 



It should be pointed out that these measurements refer to the rate- 

 limiting intermediate in the reaction sequence. Fortunately, this inter- 

 mediate has the largest steady-state concentration and is most readily 

 detected by physical methods in the case of peroxidase and catalase. In 

 this case, equation 5 applies to the rate of combination of enzyme and 



