A novel spectrophotometric assay for lipase activity utilizingcis-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 usescis-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 μM PnA, 1 μM BSA, 75 μM triolein, and 0.3 mM taurocholate in a 50 mM tris-HCl buffer with 1 μM 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 forCandida cylindracea lipase obtained with this assay are similar to those obtained with¹⁴C-labelled triolein.     

Hydrolysis of fluorescent pyrene-acyl esters by human pancreatic carboxylic ester hydrolase and bile salt-stimulated lipase

Fluorescent esters containing pyrenedecanoic acid (P₁₀) or pyrenebutanoic (P₄) acid (P₄cholesterol, P₁₀cholesterol, P₄- and P₁₀-containing triacylglycerols) were synthesized and used as substrates for human pancreatic carboxylic ester hydrolase and bile salt-stimulated lipase from human milk. Both enzymes were purified by immunoaffinity chromatography. All fluorescent pyrene derivatives were hydrolyzed by pancreatic carboxylic ester hydrolase and bile salt-stimulated lipase, but at different rates. The hydrolytic rates of the “short” acyl esters (P₄-containing esters) were higher than those of the “long” ones (P₁₀-containing esters). Conditions were optimized for sensitivity of the assay using fluorescent cholesteryl esters. The pH optimum was 7.5–8.0. Sodium cholate exhibited a stronger activating effect than taurocholate or taurodeoxycholate (maximal activation was achieved with 5 mmol/L cholate and with a molar ratio cholesteryl ester/cholate around 1∶10). Both pancreatic carboxylic ester hydrolase and bile salt-stimulated lipase from milk were strongly inhibited by the other amphiphiles tested, namely phosphatidylcholine and Triton X-100, and were inactivated by low concentrations (10 μmol/L) of the serine-reactive diethyl-paranitrophenyl phosphate (E₆₀₀). Both enzymes were strongly inhibited by relatively low concentrations of plasma low density lipoproteins. These studies indicate that the fluorescent esters containing pyrene fatty acids can be used as substrates for assaying and investigating the properties of pancreatic carboxylic ester hydrolase as well as bile salt-stimulated lipase from milk.     

An NMR assay for quantitating lipase activity in biphasic macroemulsions

A novel nuclear magnetic resonance (NMR) assay has been developed to monitor lipase-catalyzed esterification reactions without the need to extract and purify the individual components. The technique measures ratios of ester:alcohol signals and has been shown to be both efficient and reproducible. The assay has proven useful as a quick screen for the effect of varying conditions on the extent of esterification in various biphasic solvent systems and can be applied to both saturated and unsaturated long-chain fatty acid and alcohol substrates. The NMR ratio technique has been used to quantitate the extent of reaction in theCandida cylindracea lipase-catalyzed synthesis of oleyl palmitate, stearyl palmitate, oleyl oleate, oleylgamma-linolenate and oleyl linoleate. The identity of these products was confirmed by high-resolution mass spectrometry.     

Lipid-lipase interactions. 2. A new method for the assay of lipase activity

A simplified procedure for the measurement of activity was developed for lipases (EC 3.1.1.3) fromChromobacterium viscosum, Candida rugosa, Aspergillus niger andRhizopus arrhizus. It differs from existing procedures in that olive oil, refined with a bleaching clay, was used as the substrate, periodic sonication was applied to promote efficient emulsification, the hydrolysis time was extended to 1 hr, and no additive such as protective colloids and surfactants were used. The activity was determined arbitrarily for that amount of lipase required to bring about 24% hydrolysis/hr at room temperature Unlike currently used assay procedures, the present method gives results which are highly reproducible with a maximum standard deviation of 2.5% and, more commonly, less than 1.00%.     

A convenient spectrophotometric assay for phospholipase D usingp-nitrophenyl-phosphocholine as substrate

A simple assay for plant phospholipase D is described. The enzyme hydrolyzesp-nitrophenyl-phosphocholine top-nitrophenyl phosphate, which in the presence of an acid phosphatase generatesp-nitrophenol (a chromogenic compound). Thus,p-nitrophenylphosphocholine can be used for assay of phospholipase D in sources that do not also contain high levels of phospholipase C. This work was supported by a U.S. Public Health Service Research Grant (GM15053).     

A colorimetric assay of pancreatic lipase: Rapid detection of lipase and colipase separated by gel filtration

A rapid assay for pancreatic lipase (E.C., glycerol-ester hydrolase 3.1.1.3) is described. The assay is based on the color change of a pH indicator as butyric acid is released from the substrate tributyrin. A mixture made with tributyrin and the water soluble components of the assay is ideally suited for use as a rapid test as, for example, when assaying chromatography fractions. Quantitative data can be obtained by measuring the disappearance of absorbance at 557 nm versus a blank reaction. The assay has been used in the rapid preparation of colipase-free lipase and colipase.     

4-取代氨基喹唑啉的简便合成

以2-氨基苯甲腈为原料,DMF为反应试剂和溶剂,在苯磺酰氯的存在下高产率地合成了中间体亚胺啶的盐酸盐,该盐酸盐用NaOH溶液处理以后得到固态中间体亚胺啶,此中间体不须纯化,直接在乙酸中与乙醇胺或苯胺反应较高产率合成了6种标题化合物,并用IR、1HNMR和MS进行了表征。     

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°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 dm^?3 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 V_max and K_m were 57·5 μmol 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.     

Hydrolysis of synthetic triacylglycerols by pancreatic and lipoprotein lipase

The stereochemical course of the hydrolysis of synthetic sn-glycerol-1-palmitate-2-oleate-3-linoleate, sn-glycerol-1,2-dipalmitate-3-oleate and their antipodes by pancreatic and milk lipoprotein lipase was investigated by thin layer and gas liquid chromatographies of the diacylglycerol intermediates. The enzymic hydrolyses were made with bile salts or lysolecithin in a 1∶1 molar ratio to the substrate as emulsifiers and were limited to short time intervals which minimized isomerization and the reversal of lipolysis. In all instances, the products of hydrolysis by lipoprotein lipase contained a marked preponderance of the 2,3-diacylglycerols, while the composition of the diacylglycerol intermediates in the products of pancreatic lipase varied with the nature of the fatty acid in the 1 and 3 positions of the triacylglycerol molecule. Pancreatic lipase, but not lipoprotein lipase, gave a preferential release of unsaturated fatty acids. The above results are similar to those obtained with radioactive trioleoylglycerol and conventional stereospecific analyses and suggest that lipoprotein lipase may favor attack on the sn-1 position. It is hypothesized that the small amounts of the 1,2-diacylglycerols present may have arisen from a reversal of lipolysis also catalyzed by this enzyme. Presented in part at the AOCS Fall Meeting, Ottawa, September 1972.     

Use of a fluorescent radiolabeled triacylglycerol as a substrate for lipoprotein lipase and hepatic triglyceride lipase

A fluorescent radiolabeled triacylglycerol has been synthesized by using a fluorescent fatty acid (pyrene decanoic acid) and a radiolabeled oleic acid. This analog of the natural substrate, 1(3)pyrene decanoic-2,3(1,2)-dioleoyl-sn-glycerol, has been tested as substrate for determining lipoprotein lipase and hepatic triacylglycerol lipase activities in post-heparin plasma. Optimal conditions for the determination of the two post-heparin plasma lipases were similar to those using radiolabeled triolein. Using this substrate, both post-heparin lipases exhibited their characteristic properties (pH optimum and effect of inhibitors) and attacked external ester bonds (1 or 3) containing pyrene decanoic and oleic acids at a similar rate     

Hydrolysis of intralipid by pancreatic lipase and phospholipase A2-gel filtration studies

Intralipid was incubated with pancreatic lipase (EC 3.1.1.3) and/or phospholipase A₂ (EC 3.1.1.4) at two bile salts/phosphatidylcholine molar ratios and at two different triglyceride hydrolysis rates using various amounts of lipase. Incubations were studied by gel filtration. Results show: (i) During lipase action, three phases of lipids coexist: an emulsified phase, a micellar phase and an intermediate heavy phase sized between the two others. The equilibrium between each phase is dependent upon the bile salts concentration. (ii) Under these conditions, pancreatic lipase was at 60% bound to the emulsified phase, whereas pancreatic phospholipase A₂ was bound at 94% to the micellar phase.     

An improved method for the colorimetric assay of lipase activity using an optically clear medium

Lipase activity can be spectrophometrically measured in an optically clear medium using long chain fatty thioesters of 1-mercapto-2,3-propanediol or 2-mercaptoethanol as substrates. With hexamethylphosphoric triamide solutions of these thiosubstrates, the Michaëlis-Menten constants of lipase fromRhizopus arrhizus were determined. The effects of calcium chloride and of bovine serum albumin on the enzyme activity were established.     

A sensitive Schiff-base fluorescent indicator for the detection of Zn2+

A Schiff-based fluorescence probe benzene-1,2-dicarbaldehyde bis-benzoyl hydrazide (L) was designed and synthesized. Its sensing behavior toward metal ions has been investigated by absorption and fluorescence spectroscopy. Indicator L showed high selectivity to Zn²⁺ in various solvents, whereas other metal ions failed to induce response. Especially Cd²⁺, which has similar chemical properties with Zn²⁺, can be distinguished from Zn²⁺ obviously.     

A fluorescent intercalator displacement assay for establishing DNA binding selectivity and affinity

A summary of the qualitative and quantitative elements of a fluorescent intercalator displacement (FID) assay useful for establishing the DNA binding selectivity, affinity, stoichiometry, and binding site size and distinguishing modes of DNA binding is provided.     

A simple and rapid colorimetric method for determination of free fatty acids for lipase assay

A simple and rapid colorimetric method was developed to determine the lipase activity for fat splitting. Free fatty acids produced by lipase from triacylglycerols were determined by observing the color developed using cupric acetate-pyridine as a color developing reagent. This modified method requires only a few minutes to determine the free fatty acids, whereas it takes over 20 min by the conventional methods which require solvent evaporation and centrifugation steps. The sensitivity and reproducibility of the method were good for caproic, caprylic, capric, lauric, myristic, palmitic, stearic and oleic acids.     

A sensitive and selective near-infrared fluorescent probe for mercuric ions and its biological imaging applications

A new mercury(II) near-infrared region fluorescent probe 3,9-dithia-6-monoazaundecane-tricarbocyanine has been designed and synthesized. It consists of two functional moieties: the tricarbocyanine performs as the near-infrared region fluorophore, and the 3,9-dithia-6-monoazaundecane acts as the selected binding site for metal ions. The near-IR excitation and emission profiles of the probe can minimize cell and tissue damage and avoid native fluorescence from natural cellular species. It exhibits fluorescence increase upon the binding of the Hg(2+) based on the inhibition of the photoinduced electron transfer quenching mechanism. Excellent sensitivity and selectivity for mercuric ions are observed with this probe. The value of the system is demonstrated by its use in monitoring the real-time uptake of Hg(2+) within HepG2 cells and five day old zebrafish. The synthesis and remarkable properties of it help to extend the development of metal ions fluorescent probes for biological applications.     

Hydrolysis of copolyesters containing aromatic and aliphatic ester blocks by lipase

Copolyesters (CPEs) prepared by the transesterification reaction between aromatic and aliphatic polyesters were hydrolyzed by Rhizopus delemar lipase. The susceptibility of CPEs to hydrolysis by this lipase dropped off rapidly during the initial stage of the transesterification reaction and increased gradually as the reaction proceeded. The susceptibility to hydrolysis decreased with increase in aromatic polyester content. It was concluded that the rigidity of the aromatic ring in the CPE chains strongly influenced their susceptibility to hydrolysis by this lipase.     

A convenient scheme for synthesizing reduction‐sensitive chitosan‐based amphiphilic copolymers for drug delivery

A novel type of reduction‐sensitive graft copolymers, chitosan‐S‐S‐poly(ε‐caprolactone) (CS‐S‐S‐PCL, here ‐S‐S‐ means PCL was conjugated onto chitosan backbone through disulfide linkage), was synthesized through a convenient route using dithiodipropionic anhydride (DTDPA) as a disulfide donor. Reaction of hydroxy‐terminated poly(ε‐caprolactone) (PCL) with DTDPA quantitatively yielded DTDPA functionalized PCL (PCL‐S‐S‐COOH). The disulfide‐containing polyester was regioselectively conjugated onto the hydroxy groups of chitosan under mild and homogeneous conditions, utilizing dodecyl sulfate‐chitosan complexes (SCC) as an intermediate. The self‐assembly and Doxorubicin (Dox) release behavior of the copolymers were investigated. Spherical micelles could be formed through self‐assembly of CS‐S‐S‐PCL in aqueous media. The reduction‐sensitive behavior of CS‐S‐S‐PCL micelles was investigated by using Dithiothreitol (DTT) as a reductive reagent. In the presence of 10 mM DTT, the micelles gradually lost their aggregation stability and were precipitated out after four days. In addition, the Dox release was accelerated when the micelles were treated with DTT. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012