Biologic and biochemical features of AHSP, a molecular chaperone for a globin. (A) AHSP prevents oxidant-induced α hemoglobin precipitation in solution. Recombinant human AHSP was preincubated with purified oxygenated (oxy) α globin at the indicated molar ratios for 60 minutes. Potassium ferricyanide (50 μM final concentration) was added at time 0 to induce heme oxidation. Protein precipitation was monitored by light scattering at 700 nm. Reproduced from Kihm et al.⁹⁷  (B) AHSP-α hemoglobin interactions in mammalian cells. COS cells expressing HA-α hemoglobin and V5-AHSP, either separately or in combination, were stained with antibodies against V5 and HA and analyzed by indirect immunofluorescence. Original magnification ×400. Reproduced from Kihm et al.⁹⁷  (C) Ahsp^−/− erythrocytes exhibit abnormal morphology and hemoglobin precipitates (Heinz bodies). Wright-Giemsa stains of mutant erythrocytes (top panels) show morphologic abnormalities and inclusion bodies, designated by * and magnified in the inset. Heinz body stain, which detects denatured globin chains (bottom panels), is positive in Ahsp^−/− cells. Photographs were taken using an Axioskop 2 microscope equipped with an Axiocam digital camera (Carl Zeiss). Images were processed using Axiovision Product Suite (Carl Zeiss) and Photoshop (Adobe, San Jose, CA). Original magnifications: panel B, 1000×; panel C, 400×. From Kong et al.⁹⁸  (D) Structure of oxidized (FeIII) holo α globin bound to AHSP. α globin and AHSP are colored yellow and blue, respectively. The heme group is shown in green and His58 and His87 are in purple. The α globin heme iron (red sphere) is in a bis-histidyl conformation, bound by His58 and His87 (shown in purple). Adapted from Feng et al.¹⁰²