JBC, Vol. 250, Issue 16, 6439-6444, Aug, 1975
Interaction of rabbit hemopexin with rose bengal and photooxidation of the rose bengal-hemopexin complex
V. L. Seery, W. T. Morgan and U. Muller-Eberhard
Rabbit hemopexin associates with rose bengal producing a hypochromic shift
in the absorption spectrum of the dye; the extinction coefficient of the
dye bound to heme-saturated hemopexin is approximately 20% lower than that
of the dye bound to the apoprotein. The interaction of apo- and
heme-saturated hemopexin with rose bengal was studied in detail by
difference spectroscopy. Apo-hemopexin has one tight binding site for the
dye with a dissociation constant in the micromolar range and a set of
several weaker binding sites. In contrast, heme-saturated hemopexin has a
very low affinity for the dye. Evidence that histidine residues of
hemopexin participate in the binding of heme was obtained by photooxidation
of hemopexin sensitized by rose bengal. Progressive modification of the 16
histidine residues of hemopexin is effected by illumination of the
dye-hemopexin complexes. The midpoint of this pH-dependent reaction is at
pH 6.8 +/- 0.1. In 15 min of irradiation, apo-hemopexin loses 50% of its
ability to form a low spin hemichrome complex with deuteroheme while only
10% of the ligand coordination to heme iron of the deuteroheme-hemopexin is
lost. At that time, approximately 2 more histidine residues are modified in
apo-hemopexin than in deuteroheme-hemopexin, and no change is found in
other potentially photolabile amino acid residues. The characteristic
circular dichroism positive extremum at 231 nm of hemopexin also was
decreased by photooxidation, and the loss was slower in the
deuteroheme-hemopexin complex than in the apoprotein. When deuteroporphyrin
IX was used as the photosensitizing agent, similar results were obtained.
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