R. L. AIRTH 



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CO 



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60 •■ 



40 ■• 



20 ■• 



30 



60 



90 120 



TIME-SECONDS 

 Fig. 1. In vitro fungal Ijiohiniinescence reaction. Lncifeiasc (homogenate — see 

 text) 2.7 mg; lucit'erin,' 0.20 ml; ITXH, 0.(35 ^nioles; 0.0.5 M K-l'O, buffer, pH 7.4, 

 containing 10— '' M Ver.sene brought to a final volinne of 3.0 nil. 



of light A\ith no further additions, this capacity is quickly lost. The 

 explanation for the rather slow increase in light intensity during 

 the course of the reaction is open to several interpretations. 



When first isolated, fimgal luciferase was reported as quite stable; 

 it could be repeatedly frozen and thawed with very little loss of ac- 

 tivity (1) . It is now known that this is the case only when very 

 crude honiogenates are used and that on further purification the 

 enzyme appears to become quite unstable. The enzyme is now pre- 

 pared by making a homogenate of an air-dried, surface-grown mycelia 

 mat Avith 0.05 M potassium phosphate buffer, pH 7.45, the buffer con- 

 taining 0.001 M Versene and 0.001 M mercaptoethanol. It is of in- 

 terest that liquid cultures grown with any form of agitation do not 

 yield luminous mycelia. Attempts to demonstrate a deficiency of 

 enzyme or substrate under such conditions have been inconclusive to 

 date. The homogenate is treated with protamine sulfate and sub- 

 sequently fractionated with (NH4) 0SO4. This procedure has re- 

 sulted in approximately a 5-fold purification in the 30% to 50% 

 (NH4) 0SO4 fraction. This increase in activity refers to both the 

 initial rate of the light-emitting reaction and the maximum value 

 finally attained. This preparation loses approximately 75% of its 

 activity on freezing and thawing once. Storage at room temperature 

 or at 35°C results in complete loss of activity in 12 hours. 



