A new method for determination of microsomal haem oxygenase (EC 1.14.99.3) based on quantitation of carbon monoxide formation.
Microsomal haem oxygenase (MHO) catalyses the main pathway for haem degradation. Hitherto bilirubin formation rate (BFR) has been used for determination of MHO activity. In the present study a method is described where MHO activity is assessed from the rate of carbon monoxide formation (VCO) in tissue homogenates with methaemalbumin as substrate. Formed CO is bound to haemoglobin present in the tissue homogenate. CO is measured by gas chromatography after reduction to methane. CO amounts of 0.01 nmol could be measured. This corresponded to an MHO activity of 0.05 pkat when using standard incubation mixture and 12 min incubation time. The correlation with MHO activity determined with the BFR technique was good (r = 0.94; n = 41); MHOVCO = 0.94-MHOBFR-0.08). Advantages with the VCO method are a ten-fold increase in sensitivity compared to the BFR method and independence of biliverdin reductase.
A new method for determination of microsomal haem oxygenase (EC 1.14.99.3) based on quantitation of carbon monoxide formation. Microsomal haem oxygenase (MHO) catalyses the main pathway for haem degradation. Hitherto bilirubin formation rate (BFR) has been used for determination of MHO activity. In the present study a method is described where MHO activity is assessed from the rate of carbon monoxide formation (VCO) in tissue homogenates with methaemalbumin as substrate. Formed CO is bound to haemoglobin present in the tissue homogenate. CO is measured by gas chromatography after reduction to methane. CO amounts of 0.01 nmol could be measured. This corresponded to an MHO activity of 0.05 pkat when using standard incubation mixture and 12 min incubation time. The correlation with MHO activity determined with the BFR technique was good (r = 0.94; n = 41); MHOVCO = 0.94-MHOBFR-0.08). Advantages with the VCO method are a ten-fold increase in sensitivity compared to the BFR method and independence of biliverdin reductase.