Phan A P, Ngo T T, Lenhoff H M
Appl Biochem Biotechnol. 1983 Apr;8(2):127-33. doi: 10.1007/BF02778093.
We have developed a highly sensitive and rapid spectrophotometric assay for tyrosine decarboxylase that can be applied to determining pyridoxal-5'-phosphate. In the assay, tyramine, a product of tyrosine decarboxylation, reacts with 2,4,6-trinitrobenzenesulfonic acid to give a product soluble in toluene whereas tyrosine does not. We determined the amount of tyramine produced enzymatically by reading the absorbance at 340 nm of a toluene extract of the reaction mixture. This method is capable of detecting as low as 2.9 micrograms/mL of the enzyme. Using this method, we find the Km for tyrosine decarboxylase from Streptococcus faecalis to be 3.55 X 10(-4)M. We have also developed a specific and extremely sensitive method for determining pyridoxal-5'-phosphate, a cofactor of the enzyme, by using this spectrophotometric assay with apotyrosine decarboxylase.
我们开发了一种用于酪氨酸脱羧酶的高灵敏度快速分光光度测定法，该方法可用于测定磷酸吡哆醛 -5'- 磷酸。在该测定中，酪氨酸脱羧产物酪胺与 2,4,6- 三硝基苯磺酸反应生成可溶于甲苯的产物，而酪氨酸则不会。我们通过读取反应混合物甲苯提取物在 340nm 处的吸光度来测定酶促产生的酪胺量。该方法能够检测低至 2.9 微克 / 毫升的酶。使用该方法，我们发现粪肠球菌酪氨酸脱羧酶的 Km 为 3.55×10⁻⁴M。我们还通过使用这种脱辅基酪氨酸脱羧酶的分光光度测定法，开发了一种用于测定该酶的辅因子磷酸吡哆醛 -5'- 磷酸的特异性极高灵敏度的方法。
