Use of naturally fluorescent triacylglycerols from Parinari glaberrimum to detect low lipase activities from Arabidopsis thaliana seedlings.
The aim of this study was to design a convenient, specific, sensitive, and continuous lipase activity assay using natural long-chain triacylglycerols (TAGs). Oil was extracted from Parinari glaberrimum seed kernels and the purified TAGs were used as a substrate for detecting low levels of lipase activities. The purified TAGs are naturally fluorescent because more than half of the fatty acids from Parinari oil are known to contain 9,11,13, 15-octadecatetraenoic acid (parinaric acid) in its esterified form. The presence of detergents (sodium taurodeoxycholate, CHAPS, Sulfobetaine SB12, Tween 20, Brij 35, Dobanol, n-dodecylglucoside) above their critical micellar concentration dramatically increases the fluorescence of the parinaric acid released by various lipases. This increase in the fluorescence intensity is linear with time and proportional to the amount of lipase added. This new method, performed under non-oxidative conditions, was applied successfully to detecting low lipase levels in crude protein extracts from plant seeds and could be scaled down to microtiterplate measurements. Quantities as low as 0.1 ng of pure pancreatic lipase could be detected under standard conditions (pH 8). Lipase activity can also be assayed in acidic media (pH 5) using human gastric lipase. This simple and continuous assay is compatible with a high sample throughput and might be applied to detecting true lipase activities in various biological samples.
Beisson
F
F
Laboratoire de Lipolyse Enzymatique (UPR 9025 du CNRS), Institut de Biologie Structurale et Microbiologie, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Ferté
N
N
Nari
J
J
Noat
G
G
Arondel
V
V
Verger
R
R
D000693:Anaerobiosis; D017360:Arabidopsis / Q000201:enzymology*; D003902:Detergents; D006801:Humans; D007700:Kinetics; D008049:Lipase / Q000378:metabolism*; D010936:Plant Extracts / Q000737:chemistry*; D012639:Seeds / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D014280:Triglycerides / Q000378:metabolism* / Q000528:radiation effects; D014466:Ultraviolet Rays
D003902:Detergents; D010936:Plant Extracts; D014280:Triglycerides; D008049:Lipase