Comparison of the direct binding affinity of deoxyHb for wild-type and 2 mutants of cdb3. Purified wild-type cdb3 (residues 1-379), a low-affinity mutant [del(12-23)] and a high-affinity mutant [del(29-52)] were immobilized onto Affi-Gel 15 beads. Derivatized beads were equilibrated in 10 mM bis-Tris acetate buffer, pH 6.5, and excess predialyzed Hb was added before incubation for 1 hour at room temperature with gentle agitation under argon. Beads were washed 2 times with deoxygenated 35% polyethylene glycol 1500 in 10 mM bis-tris acetate buffer, pH 6.5, to remove unbound Hb. Bound Hb was then determined by stripping the beads with 100 mM trisodium phosphate, 500 mM NaCl, pH 7.4, and measuring the absorbance of the eluate at 540 nm. The molar ratio of bound Hb to cdb3 was calculated as the ratio of bound Hb tetramers to cdb3 monomers.