ROBERT A. ALBERTY 



transient-State kinetics of peroxidase, has pioneered this field of 

 enzyme kinetics. The advantage of transient-state studies over 

 steady-state studies is that the rate constants for individual steps 

 may be determined. Complete analytic solutions have not been 

 obtained for the transient periods of enzymatic reactions, but 

 experimental data may be correlated with the solution of the 

 complete set of differential equations representing the mecha- 

 nism by use of a differential analyzer. 



Since the rate of the initial reaction of enzyme and substrate 

 is so rapid, a special mixing apparatus is required for studies of 

 this phase of the reaction. Two different experimental arrange- 

 ments have been used. In the "stopped-flow" method the 

 reactants are mixed and quickly brought to rest. Then the 

 concentration of one of the reactants or intermediates is deter- 

 mined as a function of time by a physical method of analysis. 

 A rapidly responding device is required for measuring the rate. 

 In the other method, the "continuous-flow" method, the re- 

 actants are mixed and injected through a tube at constant 

 velocity. A device placed at some point along the tube is used 

 to measure the concentration of a reactant or intermediate at 

 that point. This is the concentration which is obtained at a 

 certain time after mixing. Since the composition of the solution 

 at this point in the tube is constant in time, a measuring device 

 with a rather long time constant may be used. By changing the 

 flow rate the composition of the solution may be obtained at some 

 other time after mixing. In essence enzyme is traded for time 

 in this method. Either a large amount of enzyme is required or 

 the reaction must be such that the enzyme may be recycled. 



A total of four intermediate compounds of horse-radish 

 peroxidase have been discovered spectrophotometrically by 

 Keilin and Mann, Theorell, and Chance. The strong absorp- 

 tion of peroxidase in the Soret region and the large changes in 

 extinction coefficient upon reaction with substrate have made 

 possible a detailed study of these intermediates. Chance has 

 found that the following mechanism represents the kinetic data 

 of the transient state : 



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