Development of a carbon dioxide-capture assay in microtiter plate for aspartyl-beta-hydroxylase.
CO2-capture methods have been used for assaying many decarboxylating enzymes including hydroxylation-coupled decarboxylation reactions. The traditional CO2-capture method involves performing the reaction in capped tubes and radiometric measurement of trapped 14CO2 by scintillation counting. In this report, a 14CO2-capture method in a 96-well microtiter plate format has been developed and a phosphor imaging system has been employed for sample measurement. The new assay method has been used successfully to assay aspartyl-beta-hydroxylase activity in microtiter plate format. The results obtained here compare favorably with those obtained from the traditional tube method. The method is sensitive, suitable for high throughput, and generally applicable to many CO2-releasing enzyme assays.
DuPont Pharmaceuticals Research Laboratories, DuPont Pharmaceuticals Company, Wilmington, Delaware 19880-0400, USA.
J H
JH
Zhang
R C
RC
Qi
T
T
Chen
T D
TD
Chung
A M
AM
Stern
G F
GF
Hollis
R A
RA
Copeland
K R
KR
Oldenburg
D002245:Carbon Dioxide; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000032:analysis*; D012588:Scintillation Counting; D012680:Sensitivity and Specificity
D002245:Carbon Dioxide; D006899:Mixed Function Oxygenases; C058291:aspartic acid 2-oxoglutarate-dependent dioxygenase