Fig. 1.

(A) Quantitation of HO-1 by heme titration – A heme titration was used to quantify purified soluble,human HO-1. Free heme dissolved in 0.1 N NaOH and 100 mM KPO4 containing 0.1% Triton X-100 and 0.1% sodium cholate, pH 7.4 was added to 0.1 ml of sHO-1 in 5 μM aliquots. The absorbance at 405 nm increased linearly with the addition of heme. The sHO-1 concentration is determined by the point at which the absorbance increase deviates from linearity. The concentration, shown here at the point that the two lines intersect, is calculated to be 24 uM. Dilution of the sHO-1 stock (1:4, 1:2) demonstrates the accuracy of the titration. (B) Spectrum of the sHO-1-heme complex – Following the addition of heme to the purified apoprotein, the complex was passed over a hydroxyapatite column to remove excess heme from the sample. The sHO-1-heme complex was quantified using the absorbance at 405 nm divided by the extinction coefficient of 140 mM⁻¹ cm⁻¹ at 405 nm. The absorption spectrum of sHO-1 reconstituted with heme produced a well-defined Soret band at 405 nm. In this sample the complex is calculated to have a concentration of ~ 20 μM.