Abstract
Traditionally, heme oxygenase (HO) activity in tissue preparations is determined spectrophoto-metrically by measuring the rate of bilirubin production, a two-enzyme reaction sequence. The adequacy of this method depends upon the presence of excess biliverdin reductase and the transparency of samples. In order to study the effects of inhibitors (e.g. Sn-heme, 25 uM), and inducers (e.g. CoCl2, 250 umoles/kg) of HO, the first enzyme in the sequence, independent of possible, effects on other parts of this catabolic pathway or its end product (bilirubin), measurement of HO activity directly by gas chromatography (GC) through quantitation of carbon monoxide (CO) generated during catabolism of heme to biliverdin is desirable. HO activity [pmoles CO/mg protein/min, x±SD(n)] was determined in 13,000xg supernatant of adult rat tissues.
This method is sensitive, rapid and simple, and permits HO activity measurements in tissue preparations as well as intact cells. Tissue is homogenized with KPO4, buffer, and typically 10 ul of the 13,000xg supernatant is used. The reaction (15 min) is performed in septum-sealed, CO-free reactors at 37°C in the presence of methemalbumin (0.8 mM) with and without NADPH (experimental, 1.6 mM and blank). The reaction is terminated by freezing to −80°C, and the head space gas is analyzed by GC.
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Vreman, H., Stevenson, D. DIRECT MEASUREMENT OF HEME OXYGENASE ACTIVITY BY GAS CHROMATOGRAPHY. Pediatr Res 21 (Suppl 4), 242 (1987). https://doi.org/10.1203/00006450-198704010-00451
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DOI: https://doi.org/10.1203/00006450-198704010-00451