HPLC determination of ferrochelatase activity in human liver.
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.
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003035:Cobalt / Q000494:pharmacology; D005294:Ferrochelatase / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008190:Lyases / Q000378:metabolism*; D008652:Mesoporphyrins / Q000378:metabolism; D008665:Metalloporphyrins / Q000302:isolation & purification; D011485:Protein Binding / Q000187:drug effects
D008652:Mesoporphyrins; D008665:Metalloporphyrins; D003035:Cobalt; D008190:Lyases; D005294:Ferrochelatase