HPLC determination of ferrochelatase activity in human liver |
| |
Authors: | R G Roberts |
| |
Institution: | Biochemistry Department, Bronglais General Hospital, Aberystwyth, Wales, UK. |
| |
Abstract: | A method utilizing HPLC to estimate ferrochelatase activity in human liver cells is presented. A partially purified homogenate of liver cells is incubated with mesoporphyrin IX and cobalt(II)ion. The ferrochelatase in the homogenate incorporates the cobalt(II)ion into the mesoporphyrin. After a fixed time (90 min) the porphyrins are extracted into an ethyl acetate-acetic acid mixture. The porphyrins are then separated using reversed phase HPLC with a mobile phase of methanol-acetonitrile-phosphate buffer pH 3.0 (200:60:30 v/v). The enzyme activity is estimated by measuring the rate of utilization of mesoporphyrin. The optimum pH and substrate concentration for the reaction have been determined. |
| |
Keywords: | |
|
|