Bioluminescent assay for serum adenosine deaminase with immobilized bacterial luciferase.
We describe a bioluminescence method for measuring adenosine deaminase activity in serum. The method involves use of batchwise enzyme reaction containing adenosine, alpha-ketoglutarate, glutamic dehydrogenase and NADH. The resulting solution is injected to the continuous-flow bioluminescence system. In the system, a bacterial luciferase and NAD(P)H:FMN oxidoreductase are covalently co-immobilized on Sepharose 4B. Carrier solution (pH 6.8) for bioluminescence reaction contains FMN and decanal. The continuous-flow light-emitting system, in which the reactor (flow cell packed with immobilized enzyme) is placed in front of a photomultiplier tube inside a photon counter, is versatile and simple. Concentration and response are linearly related from 1.2 to 92.5 pmol per injection of ammonia. The precision of the method is satisfactory (coefficient of variation 3.9-6.8%). We validated the technique by comparing results with conventional assay method (UV method). Normal values for adenosine deaminase activity of serum ranged from 7.0 to 22.0 U/l in agreement with those obtained by other method. The Sepharose 4B-immobilized enzymes are stable for more than one year. This assay system could be used as a routine clinical laboratory test in the diagnosis of liver damage.
Oda
K
K
Kyoto Pharmaceutical University, Japan.
Yoshida
S
S
Hirose
S
S
Takeda
T
T
D000241:Adenosine; D000243:Adenosine Deaminase / Q000097:blood*; D001798:Blood Proteins / Q000032:analysis; D004800:Enzymes, Immobilized; D038181:FMN Reductase; D005969:Glutamate Dehydrogenase; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007656:Ketoglutaric Acids; D008107:Liver Diseases / Q000201:enzymology; D008156:Luciferases; D008163:Luminescent Measurements; D009243:NAD; D009247:NADH, NADPH Oxidoreductases; D009700:Nucleoside Deaminases / Q000097:blood*; D011786:Quality Control
D001798:Blood Proteins; D004800:Enzymes, Immobilized; D007656:Ketoglutaric Acids; D009243:NAD; D008156:Luciferases; D005969:Glutamate Dehydrogenase; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases; D009700:Nucleoside Deaminases; D000243:Adenosine Deaminase; D000241:Adenosine