IV. THE KINETICS OF HEAT INACTIVATION OF CYPRIDINA LUCIFERASE

Aurin M. Chase ¹

¹ From the Physiological Laboratories, Princeton University, New Jersey, and the Marine Biological Laboratory, Woods Hole, Massachusetts

Solutions of the enzyme luciferase, extracted from Cypridina, were subjected at pH 6.8 to temperatures from 40–55°C. for times up to 24 hours. After the desired exposures samples were cooled rapidly to room temperature, mixed with luciferin, and the first order velocity constants (representing luciferase activity) of the resulting luminescent reactions were determined by a photo electric method.

The form of the curve relating luciferase activity to time of exposure to a temperature in the above range is compound in nature. If the exposure to the high temperature is not too long, about two-thirds of the lost activity is slowly regained on standing at room temperature.

The data were described by an equation which represents the following mechanism:

See PDF for Equation

A plot of the logarithm of the rate constant, k₁, against the reciprocal of the absolute temperature yielded an experimental activation energy for this reaction of about 57,000 calories, typical of protein denaturation processes. Log k₂ plotted against 1/T was described by either a curve or two straight lines, high activation energies resulting in either case, again indicating protein denaturation. The plot of log k₃: vs. 1/T showed no apparent dependence upon temperature, k₃ being practically constant over the range studied. This may indicate that the underlying mechanism is not actually as simple as pictured. Two other mechanisms that were also considered were discarded because of lack of experimental support.

Measurements of the decrease of luciferase activity at 48°C. and at pH 6.7, pH 5.5, and pH 7.9 showed that inactivation of the enzyme at this temperature was much more rapid at pH 7.9 than at pH 6.7 and was even faster at pH 5.5.

These results from the Cypridina luminescent system were compared with those of other investigators on other systems.

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