P-Nitrophenyl laurate

Basic Protocol 3 Spectrophotometric Determination of Lipase Activity Using p-Nitrophenyl Laurate as Substrate Reagents and Solutions Commentary... 

Place 0.05 to 0.50 ml of 0.5 mM p-nitrophenol standard solution into ten individual 15- to 20-ml test tubes and dilute each to 5 ml with 0.1 M Tris-Cl buffer, pH 8.2. This yields a standard curve of 0.005 to 0.05 fimol p-nitrophenol/ml. 2. Measure A410 using 0.1 M Tris Cl buffer, pH 8.2, as a blank, and make a standard curve by plotting A410 versus the p-nitrophenol concentration in each tube. 3. For each lipase activity assay, place 2.5 ml of 0.1 M Tris-Cl buffer, pH 8.2, and 2.5 ml of 420 pM p-nitrophenyl laurate substrate solution into a 15- to 20-ml test tube. Prepare one extra tube for a reagent blank. 4. Add 1 ml water to the reagent blank. Lypolytic Enzymes... 

Place 0.0135 g p-nitrophenyl laurate (mol. wt. 321.4), 0.017 g sodium dodecyl sulfate (SDS), and 1.00 g Triton X-100 into a 100-ml volumetric flask and bring to volume with water. Heat the mixture in a water bath at 65°C for 15 min, mix well, and let the solution cool to ambient temperature prior to use. Store up to 3 days at 4°C. Reheat if the solution becomes turbid. 

All of the described procedures use emulsified substrate. Although the p-nitrophenyl laurate assay cocktail is stable for 3 days at 4°C, the emulsified olive oil substrates (or other triacylglycerol-based substrate systems) should be made fresh daily and rehomogenized periodically and when separation is visually evident. Use of day-old emulsion substrate will yield increased blank values for titratable acidity, and this effectively compromises the limit of detection of activity. Emulsified substrates should be in liquid form at common assay conditions (20° to 50°C), and partially solidified substrates (those rich in long-chain saturated fatty acids) will cause interfacial irregularities and confound the assessment of lipases in ways that cannot be accounted for. 

Amine p-Nitrophenyl acetate p-Nitrophenyl caproate p-Nitrophenyl laurate... 

Fig. 34. The effect of solvent composition of aqueous dioxane on the hydrolysis rate constant of p-nitrophenyl laurate. Catalyst ( ), imidazole (o), Cyclo-(D-Leu-His)...

Very recently, Imanishi and others reported that cyclo (D-Leu-L-Ifis) IJvias much more effective catalyst than imidazole, the corresponding linear peptide (D-Leu-L-His), and cyclo (L-Leu-L-His) 18 in the hydrolysis of p-nitrophenyl laurate 19 (135). The second-order rate constants (20% aqueous dioxane, 25 C) for these... 

It would be expected that hydrophobic bonding of a substrate to a polymer carrying catalytic groups could also serve to enhance the catalytic efficiency and an attempt to demonstrate such an effect has recently been described (52). It was found that when p-nitrophenyl laurate was added to polyethyleneimine partially acylated with a long-chain fatty acid, p-nitrophenol was extremely rapidly released. However, it seems likely that this reaction involves aminolysis of the ester rather than catalysis of a hydrolytic process and it remains to be seen whether true catalysis can be achieved in this manner. 

As a prelude to the discussion of reactions at micellar interfaces and in the interstices of micelles we should examine what is special about the nature of reactions at liquid-liquid interfaces. Adsorption of substances at interfaces can lead to an ordering of molecules that is not encountered in bulk solution. The topic has been reviewed by Menger [3,4] who has illustrated the possibilities of reactions at interfaces by investigation of the reaction of the water-insoluble ester p-nitrophenyl laurate in heptane with imidazole in an adjacent aqueous phase. Because of the insolubility of the ester in water any reaction between the species must occur at the interface. Migration of the reactants to the interface was partially rate-determining small amounts of laurate ion which adsorb at the interface retarded the interfacial hydrolysis of the ester. The adsorbed laurate ion probably impedes the transport of one or more of the reactants to the interface, suggesting a means of control of reactions not available in normal bulk reactions. 

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