Preparation of haemoglobin vesicles
HbVs were prepared as previously described16,17,23.
Briefly, Hb was purified from outdated donor human blood provided by the
Japanese Red Cross Society. The encapsulated carbonylhemoglobin (HbCO,
38 g/dL) contained 5.9 mM pyridoxal 5’-phosphate (Sigma Chemical, Saint
Louis, USA) as an allosteric effector for regulating oxygen affinity.
The lipid bilayer was a mixture of
1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine; cholesterol; and
1,5-bis-O-hexadecyl-N-succinyl-L-glutamate at a molar ratio of 5:4:0.9
and 1,2-distearoyl-sn-glycero-3-phosphatidyl-ethanolamine-N-PEG (0.3
mol%). HbV particles were suspended in a physiological salt solution.
Nitrogen gas was bubbled through the solution for storage. The
properties of HbV used in this experiment are shown in Table S1. HbV was
sufficiently retained in the blood (half-life: 47-72
hours)24. Before the experiments, the HbV solution was
mixed with a 25% human serum albumin (Benesis, Osaka, Japan) to adjust
the albumin concentration of the vesicle-suspension medium to 5 g/dL.
Under these conditions, the colloid osmotic pressure of the suspension
was maintained stable at approximately 20 mmHg24. HbVs
were stored in deoxygenated glass vials at 4°C for at least 10-12 months
and used for all experiments as the HbV test solution.