Spectrophotometric Assessment of Heme Oxygenase-1 Activity in Leishmania-infected Macrophages

Background

Reactive oxygen species (ROS) is one of the major host defense arsenals against invading pathogens used by macrophages (Missall et al., 2004). On the other hand, intra-macrophage pathogens neutralize early oxidative burst for their successful persistence within macrophages (Paiva and Bozza, 2014). In response to such oxidative stress, organisms can deploy antioxidant enzymes of host cells such as superoxide dismutase (SOD), catalase (CAT) glutathione peroxidase (GPX), and heme oxygenase-1 (HO-1) to scavenge ROS (Kathirvel et al., 2010). Intracellular parasite Leishmania donovani could effectively exploit host antioxidant enzyme HO-1 for ROS neutralization (Saha et al., 2019). HO-1 is a potent anti-oxidant enzyme catalysing the oxidative cleavage of heme to generate carbon monoxide (CO), ferrous iron (Fe²⁺), and biliverdin (BV). The biliverdin is further acted upon by another enzyme biliverdin reductase (BVR) to produce bilirubin (BL) (Tenhunen et al., 1969).There are several techniques to quantify the activity of HO-1 based on detection of one of its ultimate reaction product bilirubin via high-performance liquid chromatography (Lincoln et al., 1988; Ryter et al., 1999), visible spectrophotometry (Schacter, 1978; Tenhunen et al., 1969) and radiochemical methods (Sierra and Nutter, 1992). Detection of CO by gas chromatography (Vreman and Stevenson, 1988) has also been used to assay HO-1 activity, but because of its complexity and subsequent product analysis steps the protocol is not variedly applicable. Now, biliverdin is the primary metabolite of the heme degradation by HO-1. However, it has poor spectral properties with an extinction coefficient (ε) of ~8 to 10 mM^-1 cm^-1 (Kutty and Maines, 1981). Thus, most common HO-1 activity assays rely on the reduction of biliverdin to bilirubin. Original spectrophotometric quantification of bilirubin for detection of HO-1 activity was outlined by Tenhunen et al. (1969). In the method, bilirubin formation was monitored spectrophotometrically by the increase in absorbance at 468 nm (ε₄₆₈ = 43.5 mM^-1 cm^-1), which is approximately 5-fold higher than that of biliverdin. Modifications of this main spectrophotometric assay for assessment of HO-1 activity was carried out and HO-1 activity was determined by monitoring bilirubin formation using the difference in absorbance at 464 to 530 nm (ε_464-530 = 40 mM^-1 cm^-1) (Maines, 1996; Maines and Kappas, 1974).The current protocol (Figure 1) utilises the same principle but are performed with certain minor modifications to make it much more convenient.