3.6. Titration of Plasma HPX and rhHPX with Heme

Titration of each apo-protein with heme was performed using 19 μM rhHPX (or plasma HPX) samples in PBS. Calculated aliquots of heme stock solution in DMSO (0.7 mM—for low L/P range, and 1.4 mM—for higher L/P range) were added stepwise to 1.5 mL of each protein solution in a 10 mm pathlength quartz cuvette. Each solution was gently mixed (by turning the cuvette upside-down), followed by UV/Vis and CD monitoring until no significant spectral changes indicate a completion of the protein/heme interactions. The complementary CD and UV/Vis spectra were collected (for each L/P step), followed by addition of the next heme increment, and so on. During titration, the samples were maintained at 20 ± 1 °C, protected from light, and flashed by argon. Initial apo-protein stock solutions served as a blank in spectral measurements. A total amount of DMSO in the titration samples did not exceed 4% v/v.