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vasmy铜虫 (小有名气)
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[求助]
求助这篇文章的酶活性是怎么计算出来的？
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他文章的单位是酶活性是 umols FFA/ mL saple. hour 测出来是吸光度OD值 他是如何计算的？ 求大神讲讲详细过程 文章原文在附件 Lipases Activity Total SN1-specific phospolipase activity was determined using (1-decanoylthio-1-deoxy-2-decanoyl-sn-glycero-3-phosphoryl) ethylene glycol (ThioPEG) as the substrate (Figure 1A).27 Hydrolysis of ThioPEG at the SN1 position produces a thiol that reacts with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) to form a mixed disulfide and the nitro-5-thiobenzoate anion. The later absorbs at 412 nm. The phospholipase activity is directly proportional to the initial velocity of the hydrolysis reaction at 37°C occurring in the wells of a 96-well plate. Initial velocity is determined by following the appearance of absorbance at 412 nm over time, for the linear portion of the curve. Briefly, an emulsion of 4.09 mmol/L ThioPEG (Avanti) in 100 mmol/L HEPES, pH 8.3, and 7 mmol/L Triton X-100 was prepared by sonication with a Bransom Sonifier. A solution of 271 mmol/L DTNB (Sigma) in dimethylsulfoxide was prepared by vortexing. A 1:1 mol mixture of ThioPEG to DTNB was prepared by adding the DTNB solution to the ThioPEG emulsion, resulting in a chromogenic substrate solution containing 4.03 mmol/L ThioPEG and 4.03 mmol/L DTNB. Total SN1 phospholipase activity was measured by adding 20 μL of a one-tenth dilution of PHP and 80 μL of chromogenic substrate to the wells of a 96-well plate and following the absorbance at 412 nm for 30 minutes in a Molecular Devices SpectraMax 250 microplate reader. The total SN1 phospholipase activity was linear between 0.5% and 10% plasma (Figure 1B). To determine the HL phospholipase activity, the PHP dilutions were preincubated in ice for 15 minutes with 1 mol/L NaCl to inhibit the EL activity. Then, the chromogenic substrate solution was added, and the plate was read at 412 nm as described previously. Finally, EL activity is calculated as the difference between total phospholipase activity and hepatic phospholipase activity. The intraassay CV for total lipase, HL, and EL were 3.8%, 3.1%, and 15.7%, respectively. The interassay CV for total lipase, HL, and EL were 3.8%, 5.3% and 28.8%, respectively. The EL CV is higher because it is the difference between total lipase and HL. In some cases, medium from monkey kidney fibroblast cell line (COS) cells overexpressing EL, HL, or LPL was used as a source of lipase.11 All enzyme activities are calculated using the molar adsorption coefficient value for the nitro-5-thiobenzoate anion, the depth of solution, and the volume of the enzyme source. Results are expressed in μmol of fatty acid released per milliliter of PHP per hour. |
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2017-02-28 10:07:32, 876.58 K
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