Francis J E, Smith A G
Anal Biochem. 1984 May 1;138(2):404-10. doi: 10.1016/0003-2697(84)90829-7.
A method for the estimation of hepatic uroporphyrinogen decarboxylase activity employing reverse-phase HPLC is described. Mouse liver homogenate in 0.25 M sucrose was pretreated with a suspension of cellulose phosphate and then centrifuged to remove hemoglobin and debris. The supernatant was used as the enzyme source. Incubations were acidified, oxidized, and centrifuged only before analysis of the porphyrins formed, using a Spherisorb ODS column and a gradient solvent system constructed from methanol/lithium citrate mixtures. Coproporphyrinogen formation by BALB/c mouse liver supernatant was estimated as about 5.0 and 9.1 pmol/min/mg protein from uroporphyrinogens I and III, respectively, at 10 microM substrate concentration and pH 6.8. Decarboxylation of pentacarboxyporphyrinogens (the last step in coproporphyrinogen formation) proved to be easily measured. Coproporphyrinogen formation from pentacarboxyporphyrinogen III abd (20 microM) at pH 6.8 was about 109 pmol/min/mg protein. Pentacarboxyporphyrinogen I was not as good a substrate as III abd but was decarboxylated faster at pH 5.4 than at 6.8, and at the lower pH and at 10 microM concentration of substrate 42 pmol of coproporphyrinogen was formed/min/mg protein. These results compared favorably with those obtained by previously published procedures involving time-consuming extraction and esterification steps.
描述了一种采用反相高效液相色谱法估算肝脏尿卟啉原脱羧酶活性的方法。将小鼠肝脏匀浆于0.25M蔗糖中，用磷酸纤维素悬浮液预处理，然后离心以去除血红蛋白和碎片。上清液用作酶源。仅在分析所形成的卟啉之前，将孵育液酸化、氧化并离心，使用Spherisorb ODS柱和由甲醇/柠檬酸锂混合物构成的梯度溶剂系统。在10μM底物浓度和pH 6.8条件下，BALB/c小鼠肝脏上清液由尿卟啉原I和III形成粪卟啉原的量分别估计为约5.0和9.1 pmol/分钟/毫克蛋白质。戊羧基卟啉原的脱羧作用（粪卟啉原形成的最后一步）被证明易于测定。在pH 6.8条件下，由戊羧基卟啉原III abd（20μM）形成粪卟啉原的量约为109 pmol/分钟/毫克蛋白质。戊羧基卟啉原I不如III abd是良好的底物，但在pH 5.4时比在pH 6.8时脱羧更快，并且在较低pH和10μM底物浓度下，每分钟每毫克蛋白质形成42 pmol粪卟啉原。这些结果与先前发表的涉及耗时提取和酯化步骤的方法所获得的结果相比更具优势。
