METHYLENE BLUE‐SENSITIZED PHOTOOXIDATION OF HEMOGLOBIN: EVIDENCE FOR CROSS‐LINK FORMATION*

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Abstract
Abstract— When exposed to light of wavelengths >520nm, human hemoglobin (Hb; I mg/m/ at pH X, 10°C) in the presence of methylene blue (MB) undergoes inter‐subunit cross‐linking, as visualized by sodium dodecyl sulfate (SDS)‐gel electrophoresis. With 10μM MB and a light dose of ˜700kJ/m2, a prominent ‘dimer’ band (28,00–30,000 daltons) appears, with traces of higher mol wt components. This effect (also observed by SDS‐Sepharose chromatography) is seen with dialyzed hemolysates and with chromatographically purified Hb. The sedimentation coefficient of photooxidized Hb in 6 M guanidine hydrochloride (2.3 S) is significantly larger than that of unirradiated Hb (1.3 S), which supports the above conclusion that stable cross‐links are generated in the photoreaction. The bonds formed are presumably not disulfides, since they resist cleavage by dithiothreitol. The rate of cross‐linking is diminished by N3, histidine, and bilirubin, but enhanced by D2O, indicating that 1O2 plays a role in the reaction. Cross‐linking is minimal when photooxidation is carried out at pH 6.0. but increases steadily with pH up to 9.0, suggesting that the reaction involves residues which titrate over this pH range.