Biophysical Characterization of PEGylated-Apohemoglobin, and the Apohemoglobin-Haptoglobin Complex

Apohemoglobin (apoHb) is a promising therapeutic candidate for scavenging free heme and reducing oxidative damage during hemolysis. This study describes the production and characterization of apoHb, PEGylated apoHb (PEG-apoHb), haptoglobin (Hp), and the apoHb-Hp complex. ApoHb was produced by using tangential flow filtration (TFF), yielding 3.47 ± 0.70 g per batch with 71.9 ± 7.1% activity. Surface PEGylation of apoHb improved its colloidal stability but reduced activity (i.e., heme-binding capacity) by ∼30%. Hp, isolated from Cohn fraction IV, formed a stable complex with apoHb, enhancing its colloidal stability, and maintained the ability to bind hemoglobin (Hb). SEC-HPLC revealed that Hp binds equivalent amounts of Hb and apoHb, and when apoHb is complexed to Hp, Hp is still able to bind an equivalent amount of Hb despite being previously bound to apoHb. Kinetic studies showed that apoHb, PEG-apoHb, and apoHb-Hp bound heme and the analogue DCNh at similar rates. PEG-apoHb significantly reduced the kinetics of Hp-binding, while apoHb-Hp bound to Hb at similar kinetic rates compared to Hp. Heme release to hemopexin (Hpx) was fastest for apoHb, slower for PEG-apoHb, and slowest for apoHb-Hp, indicating an enhanced heme retention by the apoHb-Hp complex. Taken together, these results highlight the potential of PEG-apoHb and the apoHb-Hp complex to serve as therapeutic agents to neutralize free heme and Hb under hemolytic conditions.