Levine R A, Pollard H B, Kuhn D M
Anal Biochem. 1984 Nov 15;143(1):205-8. doi: 10.1016/0003-2697(84)90577-3.
Tyrosine hydroxylase can be measured by release of tritiated water from labeled tyrosine, and the assay method has now been modified to allow recovery of 3H2O from the reaction mixture in a much more rapid and less tedious manner than previously possible. In the new method, the tyrosine hydroxylase reaction is stopped with sodium carbonate, pH 11.6. At this pH the tritium in 3H2O, but not other 3H species, is extracted into an organic scintillant containing 25% isoamyl alcohol, toluene, 2,5-diphenyloxazole, and p-bis-[2-(5-phenyloxazolyl)]benzene. The selective extraction occurs by means of exchange of tritium in 3H2O with the hydroxyl proton of isoamyl alcohol. It is the [3H]isoamyl alcohol that is then extracted into the scintillant and quantified by liquid scintillation spectrometry. Although the organic extraction method is somewhat less sensitive than the more frequently used ion-exchange method for isolating the 3H2O formed in the tyrosine hydroxylase reaction, it is much more rapid, as well as cost effective, since the enzyme reaction, extraction, and counting are carried out within the same vial.
酪氨酸羟化酶可以通过标记酪氨酸释放出的氚化水来测定，并且现在已经对测定方法进行了改进，使得能够以比以前更快且更简便的方式从反应混合物中回收³H₂O。在新方法中，用pH 11.6的碳酸钠终止酪氨酸羟化酶反应。在此pH值下，³H₂O中的氚（但不是其他³H物种）被萃取到含有25%异戊醇、甲苯、2,5-二苯基恶唑和对双-[2-(5-苯基恶唑基)]苯的有机闪烁剂中。选择性萃取是通过³H₂O中的氚与异戊醇的羟基质子进行交换来实现的。然后将[³H]异戊醇萃取到闪烁剂中，并通过液体闪烁光谱法进行定量。尽管有机萃取法在分离酪氨酸羟化酶反应中形成的³H₂O方面比更常用的离子交换法灵敏度稍低，但它速度更快，而且具有成本效益，因为酶反应、萃取和计数都在同一个小瓶中进行。
