Chemiluminescence assay of lipase activity using a synthetic substrate as proenhancer for luminol chemiluminescence reaction.
A novel chemiluminescence (CL) assay method for lipase (triacylglycerol lipase, E.C.3.1.1.3) activity was developed by using the lauric acid ester of 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI) as a substrate. The method is based on the enhanced CL reaction of luminol-hydrogen peroxide-horseradish peroxidase (HRP) with HDI that is liberated from the substrate by enzymatic hydrolysis. To simplify the assay procedure, both the hydrolysis of the substrate and the enhanced CL reaction were performed in the same reaction mixture. Lipases from Candida cylindracea and porcine pancreas were successfully determined with the detection limits (blank signal + 3 SD) of 0.05 and 50.0 mU/tube, respectively. The method is simple and rapid, permitting the completion of single assay within 5 min. The reproducibilities obtained with replicate assays were relative standard deviations (RSDs) of <=> 4.7% for within-day and <=> 6.0% for between-day assays.
Ichibangase
Tomoko
T
Graduate School of Biomedical Sciences, Course of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan.
Ohba
Yoshihito
Y
Kishikawa
Naoya
N
Nakashima
Kenichiro
K
Kuroda
Naotaka
N
D000818:Animals; D002175:Candida / Q000201:enzymology; D006735:Horseradish Peroxidase / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D006868:Hydrolysis; D007093:Imidazoles / Q000737:chemistry*; D008049:Lipase / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D008165:Luminol / Q000737:chemistry*; D010179:Pancreas / Q000201:enzymology; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013552:Swine; D014280:Triglycerides / Q000032:analysis / Q000378:metabolism
C498840:2-(4-hydroxyphenyl)-4,5-diphenylimidazole; D007093:Imidazoles; D014280:Triglycerides; D008165:Luminol; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D008049:Lipase