282 LIGHT AND LIFE 



luminescence, yeast concentrates, or of crude extracts from beef or 

 rat liver, heart, kidney, or spleen. After each of these additions, the 

 luminescence system would still respond to the addition of boiled 

 Renilla extract. Thus Renilla luciferin cannot be demonstrated in a 

 variety of naturally occurring materials. Similar observations have 

 also been made in this laboratory for finigal and firefly luciferins. 



Specificity of Renilla Luciferin and Luciferase 



Luciferin preparations from a number of bioluminescent systems 

 were tested for activity in the Renilla luminescence system. Table 

 2 shows that firefly, Cypridina, bacterial, or fungal luciferin will 



TABLE 2 



Effect of Various Luciferin Preparations on the 



Luminescence of Renilla Extracts 



Source of Luciferin 



Preparation Added Light Intensity 



None 



Renilla 60 



Firefly 



Cypridina 



Bacterial (FMNH,) 



Fungal 



Conditions: Same as described under assay system except that the source of luciferin 

 was that listed in the table, and the enzyme preparation was a resolved enzyme of the 

 type used to obtain the data plotted in Fig. 4. 



not replace Renilla luciferin. All luciferin preparations were shown 

 to be active in their respective luminescence systems prior to testing 

 them with Renilla luciferase. Likewise, Renilla Iticiferin will not 

 react in any of the above-mentioned luminescence systems. Renilla 

 luciferin is specific, therefore, for Renilla luciferase. 



Effect of Enzyme Concentration, pH, and Temperature 



Fig. 5 illustrates the eFect of luciferase concentration on luinines- 

 cence. A typical straight-line response is obtained with increasing 

 enzyme concentration. 



The response of this luminescence system to pH is shown in Fig. 6. 

 The activity declines on the acid side of neutrality, but shows con- 

 siderable activity over a relatively wide range on the alkaline side, 

 ^vith an optimum at pW 7.5. 



Fig. 7 illustrates the effect of tcmperatine on luminescence. Under 

 these experimental conditions, the optimum for this reaction is 32 °C. 



