38 INFLUENCE OF TEMPERATURE ON BIOLOGICAL SYSTEMS 



In a number of cases, higli accuracy of rate data is required because of 

 the small effect of temperature upon the overall rate. Catalase, for ex- 

 ample, gives values of 1500 cal. for the bacterial preparation or as low as 

 600 cal. in the case of the beef liver preparation. Over a 40° temperature 

 range, e.g. between 0° and 40°C, the corresponding change of rate is by a 

 factor of only 1.42 or 1.15, respectively. Highly accurate methods of meas- 

 urement are obviously needed for these reactions. 



Direct measurements of the rate of formation of enzyme substrate com- 

 pounds are ideal for purposes of determining a single reaction velocity 

 constant and the effect of temperature upon it. Such methods are not 

 always applicable, either because the enzyme-substrate compound is not 

 detectable by physical methods (5) or because the speed of the combina- 

 tion of the enzyme and the substrate is too rapid for kinetic measurements 

 over a reasonable range of substrate concentrations and temperatures. In 

 the case of hemoproteins, the rapid flow method has been applied success- 

 fully to the study of the formation of intermediate compounds (6) and 

 Gibson and Roughton (7) have obtained considerable data on the effect 

 of temperature on the kinetics of oxygen and hemoglobin. Only preliminary 

 thermal data are available in the case of catalase and peroxidase due to 

 the extreme rapidity with which the intermediates form and the small 

 amounts of purified material available. 



In this paper we present a brief evaluation of three methods for measur- 

 ing reaction velocity constants in enzyme reactions and the effects of 

 temperature upon them. Applications of the methods will be included 

 and will largely be based upon studies of intermediate compounds of 

 catalase and peroxidase. 



DETERMINATION OF REACTION VELOCITY CONSTANTS FROM 

 THE OVERALL REACTION 



Catalase and peroxidase kinetics give two examples of the complications 

 involved in determination of reaction velocity constants from the overall 

 reaction. In the case of peroxidase, a relatively straightforward determina- 

 tion of the reaction velocity constants defined by the equation: 



E + S ^— ^ ES 



ES + AH ^ E + SH + A 



may be obtained, provided that due regard is paid to the limitations of the 

 following equation (8) : 



dx _ e 



dt ~ J_ J_ (2) 



hta, kiX 



