原文我只想翻译分光光度计法

原文：我只想翻译分光光度计法Acetyl-CoA Carboxylase from Rat LiverEC 6.4.1.2 Acetyl-CoA: carbon-dioxide ligase (ADP-forming)By TADASHI TANABE, SHIGETADA NAKANISHI, TAKASHI HASHIMOTO, HIDEO OGIWARA, JUN-ICHI NIKAWA, and SHOSAKU NUMAATP+HCO3+acetyl-CoA ADP+Pi+malonyl-CoAAssay Methods The principles underlying the various assays of acetyl-CoA carboxylase have been described in previous articles in this series.1-4 Most conveniently, the enzyme activity is determined by 14CO2fixation assay or by the spectrophotometric assay in combination with the pyruvate kinase and lactate dehydrogenase reactions. The 14CO2fixation assay can be used for enzyme preparations from all steps, whereas the spectrophotometric assay is applicable to preparation from the DEAE-cellulose chromatography step and subsequent steps.14CO2Fixation MethodReagentsTris-HCl buffer, 0.5M, PH 7.5Potassium citrate, 0.1MMgCl2, 0.1MReduced glutathione, 0.1M, PH 7.5Bovine serum albumin, 3%ATP, 0.5MAcetyl-CoA, 10mMKH14CO3 (0.25Ci/mol), 0.2MHCl, 5MScintillator solution: 4g of 2,5-diphenyloxazole and 0.1g of 1,4-bis 2-(4-methyl-5-phenyloxazolyl)benzene in 1 liter of toluene plus o.5 liter of Triton X-100Procedure: When the crude extract is assayed, it is passed through a Sephadex G-50 column to remove endogenous substrates. Because rat liver acetyl-CoA carboxylase requires preincubation with citrate to attain its full activation,5 the enzyme is first preincubated at 37 for 300 min in a mixture containing 50mM Tris-HCl buffer, PH 7.5, 10mM potassium citrate, 10mM MgCl2, 3.75mM glutathione, and 0.75mg of bovine serum albumin per milliliter. The reaction is then initiated by adding an aliquot of the preincubated enzyme (up to 0.2mU) to an assay mixture (final volume, 0.8ml) containing 50mM Tris-HCl buffer, PH 7.5, 10mM postassium citrate, 10mM MgCl2, 3.75mM ATP, 0.125mM acetyl-CoA, and 12.5mM KH14CO3(0.25Ci/mol). After incubation at 37 for 10 min, the reaction is terminated with 0.2ml of 5M HCl. The reaction mixture is allowed to stand in a vacuum desiccator for 30 min to remove the unreacted H14CO3and is centrifuged at 1500g for 10 min to eliminate the insoluble material. A 0.5-ml aliquot of the supernatant is taken to dryness at 60 in a counting vial in a vacuum desiccator. After addition of 0.5ml of distilled water and 10 ml of the scintillator solution, the radioactivity is determined with the use of a liquid scintillation spectrometer. Under the assay conditions described the reaction follows Zero-order kinetics, and the initial rate of reaction is proportional to enzyme concentration.Spectrophotometric MethodReagentsKHCO3, 1MPotassium phosphoenolpyruvate, 40mMNADH, 5mM, PH 8Pyruvate kinase (rabbit muscle; Boehringer), 10mg/mlLactate dehydrogenase (rabbit muscle; Boehringer), 5mg/mlOther reagents, as for the 14CO2-fixation methodProcedure: The assay mixture contains 50mM Tris-HCl buffer, PH 7.5, 10mM potassium citrate, 10mM MgCl2, 3.75mM glutathione, 0.75mg of bovine serum albumin per milliliter, 3.75mM ATP, 0.125mM acetyl-CoA, 25mM KHCO3, 0.5mM potassium phosphenolpyruvate, 0.125mM NADH, 15g or pyruvate kinase and 6g of lactate dehydrogenase per milliliter, and enzyme (up to 5 mU) in a final volume of 0.8ml. A mixture (0.76ml) containing all ingredients except ATP and KHCO3 is preincubated at 37 for 10 min in a cuvette with 1cm light path. The oxidation of NADH is followed at 37 with a recording spectrophotometer at 340nm (or at 334nm). After addition of ATP, the consumption of NADH is followed for 1 min, and the reaction is then started by addition of KHCO3. Initial velocities are obtained from the initial slopes of the recorder traces. Under the assay conditions described, the reaction follows zero-order kinetics for at least 3 min, and the initial rate of reaction is proportional to enzyme concentration.分光光度计法检测ACC活性【原理】ACC Acetyl-CoA+ATP+HCO- 3 Malonyl-CoA+ADP+Pi 该催化反应过程会消耗NADH、HCO- 3、ATP，生成Malonyl-CoA、ADP等。目前检测ACC的方法有两种：1、 放射法：一般采用14C标记，放射计数检测HCO- 3转化情况，判断ACC的活性。2、 分光光度计法（本方法）：检测NADH的消耗量，判断ACC的活性。【活性单位定义】 在37的检测条件下，反应体系中每分钟催化生成1molMalonyl-CoA或ATP的酶定义为1个活性单位（1U）。【试剂】Tris-HClKHCO3NADH磷酸烯醇式丙酮酸钾柠檬酸钾MgCl2还原性谷胱甘肽牛血清白蛋白ATP乙酰CoA丙酮酸激酶乳酸脱氢酶1、 底物缓冲液Tris-HCl50mM/L PH7.5柠檬酸钾10mM/LMgCl210mM/L谷胱甘肽3.75mM/L牛血清白蛋白0.75mg/ml乙酰CoA0.125mM/L磷酸烯醇式丙酮酸钾0.5mM/LNADH0.125mM/L丙酮酸激酶15g/ml乳酸脱氢酶6g/ml2、 启动液ATP3.75mM/LKHCO325mM/L3、 待测酶液 每个反应管酶最大浓度不超过5mU。本文献为肝脏匀浆。 匀浆液用量及匀浆稀释度视具体情况而定；血清用量也要视实际情况调整。【操作步骤】空白管测定管底物缓冲液0.76ml0.76ml待测酶液（血清/匀浆） ml ml混匀，37水浴，10min启动液0.04ml0.04m 注：启动液应先预温好，以保证启动液加入后反应即可进行。迅速加入启动液混匀立即计时。【检测】 用紫外可见分光光度计在340nm处检测吸光度，石英比色杯，光径1cm；双蒸水调零。读取1min时各管的吸光度。【活性计算】1、血清：ACC（U/L）2、血浆：ACC（U/L） 考马斯亮兰法测定蛋白含量P（mg/L） ACC（U/mgpro）A340nm=A（测定管）A（空白管）为吸光度相关系数，即1mol的NADH对应的习光度值A，通过下列NADH标准曲线测得。【NADH标准曲线】0mol、50mol、75mol、100mol、125mol、150mol测定个点A值，计算而得。参考文献：Acetyl-CoA Carboxylase from Rat LiverEC 6.4.1.2 Acetyl-CoA: carbon-dioxide ligase (ADP-forming)By TADASHI TANABE, SHIGETADA NAKANISHI, TAKASHI HASHIMOTO, HIDEO OGIWARA, JUN-ICHI NIKAWA, and SHOSAKU NUMAMETHODS IN ENZYMOLOGY, 1981, VOL, 71: 5-16