Novel manufacturing method for producing apohemoglobin and its biophysical properties

Area of Science:

• Biochemistry
• Protein Chemistry
• Biophysics

Background:

• Apohemoglobin (apoHb) is a dimeric protein with vacant heme-binding sites.
• Current apoHb production methods are inefficient, difficult to scale, and use hazardous solvents.

Purpose of the Study:

• To develop a novel, scalable, and safer method for producing high-purity apoHb.
• To comprehensively analyze the biochemical and structural properties of the produced apoHb and reconstituted Hb (rHb).

Main Methods:

• Heme extraction using acidified ethanol solution.
• Tangential flow filtration for heme-apoHb separation.
• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for purity analysis.
• Size exclusion chromatography (SEC), circular dichroism (CD), mass spectrometry (MS), and reverse-phase chromatography for structural and biochemical characterization.
• Biophysical characterization of reconstituted Hb (rHb).

Main Results:

• Achieved >95% total protein and ~75% active protein yields with <1% residual heme and >99% purity.
• apoHb remained stable at various temperatures (4°C, -80°C) and in lyophilized form.
• Structural analyses confirmed apoHb is dimeric with reduced helical content compared to Hb, and subunits showed no oxidative modifications.
• apoHb bound haptoglobin similarly to native Hb.
• Functional characterization of rHb showed kinetics identical to native Hb.

Conclusions:

• A novel, scalable, and safe method for apoHb production was successfully developed.
• The produced apoHb is stable, pure, and structurally/functionally comparable to native Hb.
• The developed method and characterization of apoHb and rHb offer significant improvements for protein research and applications.