Abstract
Haptoglobin forms a stable, irreversible complex with hemoglobin. The H chain of haptoglobin, which is the subunit that binds hemoglobin, shows strong sequence homology with the serine protease family. This raises the question of whether hemoglobin binds to the protease-like active site pocket of H chain as the protease inhibitors do with serine proteases. This question can be tested by binding proflavin and thionin to haptoglobin because these dyes are known to interact specifically with serine proteases at the peptide binding site. A single, specific binding site, characteristic of the serine proteases, was found for haptoglobin with association constants for proflavin or 1.4 x 10(3) at pH 7.1 and 8.2 x 10(3) at pH 9.5 and for thionin of 3.5 x 10(3) at pH 7.1. In order to confirm that these dyes are indeed binding to the specificity pocket of haptoglobin, competition experiments with classical serine protease substrates and inhibitors were performed. The results showed that trypsin-specific substrates and inhibitors did compete with proflavin binding, as expected from the homology, and that reagents of a chymotryptic specificity did not. When the dye titrations were performed on haptoglobin-hemoglobin complex, the same binding constants were obtained as for haptoglobin alone. This demonstrates that the active site-like region of haptoglobin and the hemoglobin binding site are mutually exclusive and do not interact in any way.
Highlights
WAVELENGTH InmlM ) with trypsin (2.0 X lo-' M ) (- - -). B , the differencespectra ' at pH 9.5 (0.05 M glycine/KOH) of proflavin
This question can be tested by binding proflavin and exposed when tetramericH b separates into dimers havbeeen thionin to haptoglobin because these dyes are known implicated (5, 10)
A modelfor the H chain structure was constructed (13). It shows that the principal of haptoglobin,competition experimentswith classical structuralelements of theserine proteases,including the serine protease substrates andinhibitors were per- intrachain disulfide bridges,8 barrels, LY helices, and the active formed
Summary
M ) with trypsin (2.0 X lo-' M ) (- - -). B , the differencespectra ' at pH 9.5 (0.05 M glycine/KOH) of proflavin Whichis in the samplecell, while the absorbance of the Hbin the reference cell is constant The resultsof this titration were analyzed, as described for the Hp-proflavin titrations,yielding a K,, of 8.8 X loiM", an. This increase in wavelength over the proflavin complexes is crucial because the Hb absorbance a t 620 nm is much lower (E 4 X 10’) than at 452 nm At the wavelength maximum of the difference spectrum, though, the absorbance is low (
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