Continuous-flow bioluminescent determination of ATP in platelets using firefly luciferase immobilized on epoxy methacrylate.
Firefly luciferase was immobilized on epoxy methacrylate beads and used for a continuous-flow assay of ATP extracted from platelets. The immobilized luciferase had a half-life of 3 days at 25 degrees C; there was a 25% recovery of luciferase activity upon immobilization, and ca 50 reactors were made from 1 mg of commercial enzyme. The sensitivity of the assay was 0.3 pmol of ATP, and the response was linear between 1 and 500 pmol of ATP. The ATP content of platelets obtained with the present method correlated well with those obtained using soluble luciferase.
Carrea
G
G
Istituto di Chimica degli Ormoni, CNR, Milano, Italy.
Bovara
R
R
Girotti
S
S
Ferri
E
E
Ghini
S
S
Roda
A
A
D000255:Adenosine Triphosphate / Q000097:blood*; D000818:Animals; D001792:Blood Platelets / Q000032:analysis*; D001517:Coleoptera / Q000201:enzymology; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D006801:Humans; D020100:Hydrogels; D008156:Luciferases; D011102:Polyhydroxyethyl Methacrylate / Q000031:analogs & derivatives
D004800:Enzymes, Immobilized; D020100:Hydrogels; C000257:polyhydroxyethyl methacrylate hydrogels; D011102:Polyhydroxyethyl Methacrylate; D000255:Adenosine Triphosphate; D008156:Luciferases