A novel spectrophotometric assay for lipase activity utilizing cis-parinaric acid.
A new spectrophotometric assay for determining the activity of acylglycerol hydrolases (lipases, E.C. 3.1.1.3) was developed and optimized for yeast lipase (Candida cylindracea). Studies with porcine pancreatic lipase were also conducted and the influence of various detergents and divalent cations on the assay was evaluated. The assay uses cis-parinaric acid (PnA), a naturally occurring fatty acid that has unique spectroscopic properties, and takes advantage of the reversible binding of fatty acids to bovine serum albumin (BSA). Free PnA has an ultraviolet absorption peak at 321.2 nm. When PnA is bound to BSA, however, the peak shifts to 324.2 nm. The assay mixture contains 6 microM PnA, 1 microM BSA, 75 microM triolein, and 0.3 mM taurocholate in a 50 mM tris-HCl buffer with 1 microM EDTA. The release of oleic acid from triolein is monitored over time by measuring the ratio of optical densities (OD) at 319.0 and 329.0 nm. Initially, there is maximum binding of PnA to BSA, and the OD ratio is approximately 1.0. Upon addition of lipase, PnA is displaced from the BSA by oleic acid released from triolein, and the OD ratio increases to a maximum of about 1.8. However, when calcium is present in the reaction mixture an insoluble calcium-PnA complex forms, resulting in a progressive decrease in OD at both 319.0 and 329.0 nm. The kinetic assay described here is simple, rapid, sensitive, reproducible, inexpensive, and it can be adapted to measure the activity of a variety of calcium-independent lipases. Under similar assay conditions, activities for Candida cylindracea lipase obtained with this assay are similar to those obtained with 14C-labelled triolein.
Center for Nutrition, Meharry Medical College, Nashville, TN 37208.
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Rogel
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Stone
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Adebonojo
D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology; D002175:Candida / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D005231:Fatty Acids, Unsaturated; D008049:Lipase / Q000032:analysis* / Q000037:antagonists & inhibitors; D008274:Magnesium / Q000494:pharmacology; D010179:Pancreas / Q000201:enzymology*; D012710:Serum Albumin, Bovine; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013223:Statistics as Topic; D013552:Swine
D005231:Fatty Acids, Unsaturated; D012710:Serum Albumin, Bovine; D008049:Lipase; D008274:Magnesium; C013641:parinaric acid; D002118:Calcium