Role of β/δ101Gln in regulating the effect of temperature and allosteric effectors on oxygen affinity in woolly mammoth hemoglobin.
The oxygen affinity of woolly mammoth hemoglobin (rHb WM) is less affected by temperature change than that of Asian elephant hemoglobin (rHb AE) or human normal adult hemoglobin (Hb A). We report here a biochemical-biophysical study of Hb A, rHb AE, rHb WM, and three rHb WM mutants with amino acid substitutions at β/δ101 (β/δ101Gln→Glu, Lys, or Asp) plus a double and a triple mutant, designed to clarify the role of the β/δ101 residue. The β/δ101Gln residue is important for responding to allosteric effectors, such as phosphate, inositol hexaphosphate (IHP), and chloride. The rHb WM mutants studied generally have higher affinity for oxygen under various conditions of pH, temperature, and salt concentration, and in the presence or absence of organic phosphate, than do rHb WM, rHb AE, and Hb A. Titrations for the O2 affinity of these mutant rHbs as a function of chloride concentration indicate a lower heterotopic effect of this anion due to the replacement of β/δ101Gln in rHb WM. The alkaline Bohr effect of rHb WM and its mutants is reduced by 20-50% compared to that of Hb A and is independent of changes in temperature, in contrast to what has been observed in the hemoglobins of most mammalian species, including human. The results of our study on the temperature dependence of the O2 affinity of rHb WM and its mutant rHbs illustrate the important role of β/δ101Gln in regulating the functional properties of these hemoglobins.
Department of Biological Sciences, Carnegie Mellon University , Pittsburgh, Pennsylvania 15213, United States.
Yue
Y
Yuan
Catherine
C
Byrd
Tong-Jian
TJ
Shen
Virgil
V
Simplaceanu
Tsuey Chyi S
TC
Tam
Chien
C
Ho
D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D000818:Animals; D005973:Glutamine / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics; D056665:Mammoths; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000737:chemistry*; D010833:Phytic Acid / Q000737:chemistry; D011485:Protein Binding; D012965:Sodium Chloride / Q000737:chemistry; D013696:Temperature
D006454:Hemoglobins; D005973:Glutamine; D012965:Sodium Chloride; D010833:Phytic Acid; D010100:Oxygen