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Oxidized-form HD5 with three intramolecular disulfide bonds was purified by reverse-phase high-performance liquid chromatography by a C18 column (06526-21, Nacalai Tesque, Inc., Kyoto, Japan) in 0.1% trifluoroacetic acid with a 0-40% acetonitrile gradient developed over 50 min at a flow rate of 1 mL/min 48,54