Structural Insights into a Hemoglobin–Albumin Cluster in Aqueous Medium

A hemoglobin (Hb) wrapped covalently by three human serum albumins (HSAs) is a triangular protein cluster designed as an artificial O₂-carrier and red blood cell substitute. We report the structural insights into this Hb-HSA₃ cluster in aqueous medium revealed by 3D reconstruction based on cryogenic transmission electron microscopy (cryo-TEM) data and small-angle X-ray scattering (SAXS) measurements. Cryo-TEM observations showed individual particles with approximately 15 nm diameter in the vitrified ice layer. Subsequent image processing and 3D reconstruction proved the expected spatial arrangements of an Hb in the center and three HSAs at the periphery. SAXS measurements demonstrated the monodispersity of the Hb-HSA₃ cluster having a molecular mass of 270 kDa. The pair-distance distribution function suggested the existence of oblate-like particles with a maximum dimeter of ∼17 nm. The supramolecular 3D structure reconstructed from the SAXS intensity using an <i>ab initio</i> procedure was similar to that obtained from cryo-TEM data