Abstract
A hemoglobin hybrid (alpha 2c beta 2 Prov) in which the alpha chains have NH2-terminal residues that have been blocked specifically by carbamylation and beta chains that have been derived from hemoglobin Providence I (beta 82 Lys leads to Asn) was prepared. This derivative shows a small but significant dependence of its oxygen equilibrium curve on the concentration of chloride, phosphate, or nitrate. These data were compared with oxygen-linked anion binding to hemoglobins A and Providence, and to the carbamylated hybrid, alpha 2c beta 2, by fitting equations to the data with the use of MULTIFIT II, a nonlinear curve-fitting program. Dependence of the anion dissociation constants from deoxygenated (KD) and fully oxygenated (Ko) hemoglobins upon the mathematical model is described. The data provide further support that Val-1 (alpha) and Lys-82 (beta) of hemoglobin are major, oxygen-linked binding sites for small inorganic anions and suggested that a third oxygen-linked site may also be present.
Highlights
HaveNH2-terminalresidues that havebeenblocked found that some inorganic anions could act as very efficient by carbamylation and /3 chains that have competitive inhibitors of thisreaction (10)
The data were not sufficiently fourpositively charged side chainsoneach,L? subunit (3). precise to distinguish amongmathematical models, orto. Some of these residues have been considered as candidates determinethestoichiometry of anion bindingH. owever, for the binding site of small inorganic anions
Some model-dependent values for oxygenbamylated at Val-1 ( p ),we proposed that Lys-82 linked anion dissociation constants havealso been computed. (p) was a major, oxygen-linkedbinding site forinorganic anions (5).Direct evidence for thisproposal waslater provided by studies on the mutant hemoglobins Providence I (7) and MATERIALS AND METHODS
Summary
HaveNH2-terminalresidues that havebeenblocked found that some inorganic anions could act as very efficient by carbamylation and /3 chains that have competitive inhibitors of thisreaction (10). Oxygen-linked anion binding to hemoglobin derivatives was measured with chloride, nitrate, and phosphate, anadnalyzed by comparison with simplified mathematical models for oxy-. Some of these residues have been considered as candidates determinethestoichiometry of anion bindingH. Subsequent curve-fitting has permitted several parameters to be calcustudies excluded His-2 ( p ) ( 4 ) and His-143 (p)(6) as binding lated which provide a clearer pictureof oxygen-linked effects sites for inorganic anions. Some model-dependent values for oxygenbamylated at Val-1 ( p ) ,we proposed that Lys-82 linked anion dissociation constants havealso been computed. Some model-dependent values for oxygenbamylated at Val-1 ( p ) ,we proposed that Lys-82 linked anion dissociation constants havealso been computed. (p) was a major, oxygen-linkedbinding site forinorganic anions (5).Direct evidence for thisproposal waslater provided by studies on the mutant hemoglobins Providence I (7) and
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