Methods for Measurements of Heme Oxygenase (HO) Isoforms-Mediated Synthesis of Carbon Monoxide and HO-1 and HO-2 Proteins

As the key enzyme in heme degradation, heme oxygenase activity governs cellular heme concentration. Heme oxygenase catalyzes the conversion of heme to carbon monoxide and bilirubin with the release of iron, which can drive the synthesis of ferritin for iron sequestration (1 ,2 ). This is the major pathway of heme degradation, and it plays a critical role in the regulation of cellular heme levels (1 ). Heme functions as a prosthetic group in hemoprotein enzymes involved in endothelial-cell function—e.g., nitric oxide synthase, soluble guanylate cyclase, cytochrome P450, peroxidase, and catalase. To date, two heme oxygenase isoforms have been characterized, each encoded by a different gene (5 –5 ). Heme oxygenase-1 is expressed under basal conditions at low levels in endothelial cells (6 –11 ) and kidney, liver, and spleen, and can be induced in these and other tissues by oxidative stress, heme, cytokines, hypoxia, nitric oxide, and heavy metals (1 ,3 ,12 ,13 ). Heme oxygenase-2 is constitutively expressed in the blood vessels, endothelium, testis, and most other tissues, where its levels are relatively unaffected by factors inducing heme oxygenase-1 (1 ,3 ). All heme oxygenase isoforms are inhibited by heme analogs in which the central iron atom is replaced by tin, zinc, or chromium (1 ,3 ,14 ). Heme oxygenase-dependent metabolism of heme to biliverdin-bilirubin and carbon monoxide has been demonstrated in homogenates of several endothelial-cell types and in arteries (15 –20 ).