Fourier-transform infrared assay of bile salt-stimulated lipase activity in reversed micelles.
A Fourier-transform infrared (FT-IR) spectroscopic method has been developed for assaying the bile salt-stimulated human milk lipase (BSSL, EC3.1) catalyzed hydrolysis of triolein in AOT reversed micelles in iso-octane. At 37 degrees C in 50 mmol dm-3 AOT the molar absorbtivities for the carbonyl stretching frequencies for triolein (at 1751 cm-1) and oleic acid (at 1714 cm-1) were 1646 dm3 mol-1 cm-1 and 743 dm3 mol-1 cm-1, respectively. The rate was linearly dependent upon the concentration of enzyme in the water pool up to 10 mg cm-3 and maximum activity was observed at a ratio (w0) of [H2O]:[AOT] = 16.7. Using these conditions, and in the presence of 10 mmol dm-3 sodium taurocholate (TC), the derived Michaelis-Menten parameters Vmax and Km were 57.5 mumol min-1 mg-1 and 5.53 mmol dm-3, respectively. These results are compared with those obtained in an oil-in-water microemulsion system and are discussed in terms of the relative partitioning of the enzyme and the substrate in the aqueous and oil phases and the interfacial concentration of the substrate in the two systems.
O'Connor
C J
CJ
Department of Chemistry, University of Auckland, New Zealand.
Cleverly
D R
DR
D001647:Bile Acids and Salts / Q000494:pharmacology*; D004143:Dioctyl Sulfosuccinic Acid; D004655:Emulsions; D005260:Female; D006801:Humans; D007700:Kinetics; D008049:Lipase / Q000032:analysis*; D008823:Micelles; D008895:Milk, Human / Q000201:enzymology; D009829:Oleic Acids / Q000032:analysis; D017550:Spectroscopy, Fourier Transform Infrared; D013501:Surface-Active Agents; D014304:Triolein / Q000032:analysis; D014867:Water / Q000032:analysis
D001647:Bile Acids and Salts; D004655:Emulsions; D008823:Micelles; D009829:Oleic Acids; D013501:Surface-Active Agents; D014867:Water; D004143:Dioctyl Sulfosuccinic Acid; D014304:Triolein; D008049:Lipase