Measurement of HO activity in DMVEC cell extracts. HO activity in uninfected and KSHV-infected DMVEC cell extracts was determined by spectrophotometric measurement of bilirubin production. Cell extracts were incubated with 50 μM heme for 30 minutes at 37°C. The reaction was stopped by the addition of 2 volumes of chloroform, and bilirubin concentration was determined by comparing the difference in optical density at 464 and 530 nm. HO activity was reported as picomoles of bilirubin produced per milligram endothelial cell protein extract per hour. KSHV-infected cells (KSHV) had 2.1-fold greater HO activity relative to uninfected cells (Mock). Treatment with the HO inhibitor CrMP reduced HO activity in infected cells to the level of uninfected cells (KSHV + CrMP), whereas treatment with the noninhibitory heme analog CuMP did not reduce HO activity (KSHV + CuMP). Results were determined in triplicate (error bars indicate ± SD).