(a) Partial trypsinolysis of EGS-crosslinked HbII. HbII was crosslinked using 4.7 μL of 1 mg/mL EGS (prepared in DMSO) for one minutes at room temperature. Trypsinolysis was done using 20 μL 0.125x trypsin stock (2.5 mg/mL prepared in 25 mM HEPES pH7) for 3.5 hours at37°C. Each lane contains 20 μL of samples. (b) The effect of 1 M hydroxylamine-HCl on EGS-crosslinked HbII. The 2 M stock was prepared in sodium phosphate buffer, pH 8.5, and equal volume was added to the EGS-crosslinked HbII sample. Samples were incubated under 37°C for 6 hours with shaking. Each lane contains 35 μL of samples unless otherwise stated. (c) Crosslinking HbII using GMBS.