Simple and rapid method for the determination of coproporphyrinogen oxidase activity.
Coproporphyrinogen oxidase, the sixth enzyme in the biosynthetic heme pathway, catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX. A reversed-phase high pressure liquid chromatography method was developed to measure coproporphyrinogen oxidase enzymatic activity in rat liver. With this method, the separation, identification and quantification of coproporphyrin III (oxidized substrate) and protoporphyrin IX (oxidized product) present in the assays could be carried out with no need of derivatization and in less than 15 min. Rat and human liver coproporphyrinogen oxidase basal activities determined using this method were 0.41+/-0.05 nmol of protoporphyrin IX/h per mg of hepatic protein and 0.87+/-0.06 protoporphyrin IX/h per mg of hepatic protein, respectively. Kinetic studies showed that optimum pH for rat CPGox is 7.3, and that its activity is linear in the range of protein concentrations and incubation times assayed. The present paper describes a sensitive, specific and rapid fluorometric high performance liquid chromatography method to measure coproporphyrinogen oxidase, which could be applied to the diagnosis of human coproporphyria, and which is also suitable for the study of lead and other metal poisoning that produce alterations in this enzymatic activity.
Sorianello
E M
EM
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón II, 4to piso, Ciudad Universitaria, (1428), Buenos Aires, Argentina.
Mazzetti
M B
MB
D000328:Adult; D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003304:Coproporphyrinogen Oxidase / Q000032:analysis* / Q000378:metabolism*; D003306:Coproporphyrins / Q000032:analysis; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D011524:Protoporphyrins / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D003306:Coproporphyrins; D011524:Protoporphyrins; C026971:coproporphyrin III; C028025:protoporphyrin IX; D003304:Coproporphyrinogen Oxidase