REACTIVITY OF TYROSINE AND TRYPTOPHAN RESIDUES IN HAPTOGLOBIN

1. 1.|The number and reactivity of tyrosine and tryptophan residues of canine haptoglobin were examined. The phenolic hydroxyl ionization of tyrosine residues was studied by spectrophotometric titrations at 295 mμ in the pH range 7.0 to 13.0. Of 29 tyrosyl residues per mole of haptoglobin, 7 were in an exposed state while the rest were buried in the interior of the native molecule. The shape of the titration curve indicated that 7 exposed tyrosine residues of pKapp = 10.4, and 15 buried with pKapp = 11.3, and 7 with pKapp = 12.3 could be distinguished. 2. 2.|Essentially similar results were obtained when the reactivity to acetylation of tyrosyl residues was examined. Thus, 8 residues reacted with N-acetylimidazole under normal conditions. In the presence of denaturing agents such as 8 M urea, an additional 20 tyrosyl residues could be O-acetylated. 3. 3.|In similar types of experiments, 5 tryptophan residues were found to react with 2-hydroxy-5-nitrobenzyl bromide under mild conditions. In addition, however, 7 more residues reacted in the presence of 8 M urea. 4. 4.|Haptoglobin preparations, in which all accessible tyrosine residues had been blocked by acetylation or all tryptophan residues had been reacted with 2-hydroxy-5-nitrobenzyl bromide, were isolated and their interaction with hemoglobin was studied. The modified haptoglobins still reacted with hemoglobin; however, the complex formed did not show any peroxidatic activity characteristic for the complex between the native haptoglobin and hemoglobin. On the other hand, when a preformed haptoglobin-hemoglobin complex was subjected to acetylation of its exposed tyrosine residues under conditions comparable to those used for haptoglobin alone, about 20% of the original peroxidatic activity remained. Blockage of the exposed tryptophan residues of a preformed haptoglobin-hemoglobin complex, however, resulted in complete loss of activity. © 1969.