Figure 7. Engineering mutant forms of Mb unable to bind oxygen.

(A) Alignment of helices E and F of mouse Mb, human Mb, human α-Hb (α-Hb), and human β-Hb (β-Hb). Iwate α-Hb and Hyde Park β-Hb are naturally occurring human Hb M mutants unable to transport O₂. Mouse HF8Y Mb is one of the Mb mutants used in this study. Numbers refer to the amino acid positions occupied by the first and last residues shown within the respective protein sequence. (B) Western blot analysis of A549 cells expressing wild-type or mutant Mb using anti-FLAG antibodies. Cells were also engineered to express GFP to track metastatic dissemination. HF8Y Mb, His(F8)Tyr Mb mutant; HE7A/HF8A Mb, His(F8)Ala/His(E7)Ala double mutant. (C) Growth rate analysis of lentiviral vector–transduced cells in the presence of 2% FBS.