Spierto F W, Whitfield W, Apetz M, Hannon W H
Clin Chem. 1982 Nov;28(11):2282-5.
With phenylalanine ammonia-lyase (EC 4.3.1.5) we converted phenylalanine (Phe) and tyrosine (Tyr) to transcinnamic acid and p-coumaric acid, respectively. These were separated by "high-performance" liquid chromatography and detected at 280 nm. We measured the Phe and Tyr content of human serum by adding 100 mU of the enzyme to a 20-microL serum aliquot, mixing for 2 h at 24 degrees C, then stopping the reaction with 1 mL of cold methanol. Precipitated proteins were removed by centrifugation and the separated clear supernates were stored at -20 degrees C. For chromatographic separation, detection, and quantification, we used a system equipped with a C-18 reversed-phase column, a variable-wavelength spectrophotometer, a printer-plotter, and a microcomputer. The mobile phase was a mixture of dilute aqueous (50 g/L) acetic acid and CH3CN (80/20, by vol). CVs for specimens containing 100 mg of Phe or Tyr per liter varied from 5 to 10%. Analytical recoveries were near 100%.
我们使用苯丙氨酸解氨酶（EC 4.3.1.5）分别将苯丙氨酸（Phe）和酪氨酸（Tyr）转化为反式肉桂酸和对香豆酸。通过“高效”液相色谱法将它们分离，并在280 nm处进行检测。我们通过向20微升血清等分试样中加入100 mU的该酶，在24℃下混合2小时，然后用1毫升冷甲醇终止反应，来测定人血清中的苯丙氨酸和酪氨酸含量。通过离心去除沉淀的蛋白质，分离出的澄清上清液保存在-20℃。为了进行色谱分离、检测和定量，我们使用了一个配备C-18反相柱、可变波长分光光度计、打印绘图仪和微型计算机的系统。流动相是稀水（50 g/L）乙酸和CH3CN（体积比80/20）的混合物。每升含有100毫克苯丙氨酸或酪氨酸的标本的变异系数在5%至10%之间。分析回收率接近100%。
