An ultraviolet spectrophotometric assay for measuring lipase activity using long-chain triacyglycerols from Aleurites fordii seeds.
In this study, we designed a specific, continuous, and sensitive UV spectrophotometric lipase assay using natural triacylglycerols (TAGs) from the Aleurites fordii seed oil (tung oil). alpha-Eleostearic acid (9,11,13-cis, trans,trans-octadecatrienoic acid) is the main fatty acid component (it accounts for up to 70%) of the TAGs from tung oil. The conjugated triene present in alpha-eleostearic acid constitutes an intrinsic chromophore, which confers strong UV absorption properties on both the free fatty acid and the TAGs from tung oil. The lipase assay is based on the difference between the apparent molar extinction coefficients of the two types of alpha-eleostearic acid present, that which is esterified into TAGs and that which is released into the reaction medium. This difference is responsible for the variations in the UV absorption spectrum of the reaction medium occurring upon enzymatic TAGs hydrolysis. Using the purified lipase from Thermomyces lanuginosa (TLL) and the detergent sodium taurodeoxycholate (NaTDC, 4 mM), it was established that the most suitable method of measuring lipolysis consisted of monitoring the decrease in the OD at 292 nm, which was linear with time and proportional to the amount of lipase added. In order to be able to estimate the specific activity of TLL, we determined an apparent molar extinction coefficient of alpha-eleostearic acid (epsilon = 13,900 M(-1) cm(-1)) under the assay conditions. Amounts of pure TLL as small as 1 ng can be easily detected in the presence of 4 mM NaTDC. Interestingly, the NaTDC concentration can be decreased as far as 0.05 mM. In comparison with other well-known methods of lipase assay, the detection limit of this new method is 100-fold lower than with the pH-stat method and similar to that of a fluorescent assay recently developed at our laboratory.
Pencreac'h
Gaëlle
G
Laboratoire de Lipolyse Enzymatique (UPR 9025 du CNRS), Institut de Biologie Structurale et Microbiologie, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Graille
Jean
J
Pina
Michel
M
Verger
Robert
R
D001203:Ascomycota / Q000201:enzymology*; D001412:Bacillus subtilis / Q000201:enzymology; D019665:Euphorbiaceae / Q000737:chemistry*; D005230:Fatty Acids, Nonesterified / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008049:Lipase / Q000032:analysis* / Q000378:metabolism; D010179:Pancreas / Q000201:enzymology; D011994:Recombinant Proteins / Q000378:metabolism; D012639:Seeds / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013656:Taurocholic Acid / Q000737:chemistry / Q000378:metabolism; D014280:Triglycerides / Q000737:chemistry / Q000378:metabolism*
D005230:Fatty Acids, Nonesterified; D011994:Recombinant Proteins; D014280:Triglycerides; D013656:Taurocholic Acid; D008049:Lipase