New approaches to the preparation and application of firefly luciferase.
Allowing for the lipid nature of firefly luciferase we have developed a new method for obtaining high-activity and high-stability enzyme preparations for bioluminescent microassay. The method includes the step of differential centrifugation in presence of stabilizing additives which entails a partial purification of the enzyme and its essential stabilization likely due to the fact that luciferase retains its lipid environment which plays an important role in catalysis. The resultant luciferase preparation is stable in solution at 4 degrees C for 2-3 months and allows the detection of down to 10(-11) M ATP. A new method has been offered for luciferase immobilization on film carriers precoated with a phospholipid layer. By sorption of the enzyme on such carriers, the samples of immobilized luciferase have been obtained suitable for constructing chemiluminescent biosensors, in the form of luciferase-containing films. There are many-fold applications for detection of ATP micro-quantities.
Filippova
N Y
NY
Chemistry Department, M. V. Lomonosov Moscow State University, USSR.
Dukhovich
A F
AF
Ugarova
N N
NN
D000818:Animals; D001517:Coleoptera / Q000201:enzymology*; D004800:Enzymes, Immobilized; D008051:Lipid Bilayers; D008156:Luciferases / Q000302:isolation & purification*; D008163:Luminescent Measurements; D012995:Solubility
D004800:Enzymes, Immobilized; D008051:Lipid Bilayers; D008156:Luciferases