A recombinant human hemoglobin with asparagine-102(beta) substituted by alanine has a limiting low oxygen affinity, reduced marginally by chloride.
A recombinant (r) mutant hemoglobin (Hb) with Asn-102(beta) replaced by an Ala (N102A(beta)) has been prepared by PCR amplification of a mutagenic DNA fragment and expression of the recombinant protein in yeast. The side chain of Asn-102(beta) is part of an important region of the alpha 1 beta 2 interface that undergoes large structural changes in the transition between the deoxy and oxy conformations. Three natural mutant Hbs with neutral substitutions of Thr, Ser, or Tyr at this site have low oxygen affinities because a hydrogen bond between Asn-102(beta) and Asp-94(alpha) in normal HbA was considered to be absent in these mutants, thereby destabilizing the oxy conformation in favor of the deoxy conformation. This proposal has been tested by expression of an rHb containing alanine at position 102(beta); alanine was chosen because its methyl side chain cannot participate in hydrogen bond formation, yet it is small enough not to disrupt the subunit interface. The nature of the desired replacement was established by sequencing the entire mutated beta-globin gene as well as the tryptic peptide containing the substitution. Further characterization by SDS-PAGE, isoelectric focusing, HPLC analysis, mass spectrometry, amino acid analysis, and sequencing of the mutant tryptic peptide confirmed the purity of the rHb. Its oxygen binding curve (2.4 mM in heme) in the absence of chloride showed that it had a very low oxygen affinity with a P50 of 42 mm Hg.(ABSTRACT TRUNCATED AT 250 WORDS)
Yanase
H
H
Rockefeller University, New York, New York 10021, USA.
Manning
L R
LR
Vandegriff
K
K
Winslow
R M
RM
Manning
J M
JM
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry; D002712:Chlorides / Q000494:pharmacology*; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016296:Mutagenesis; D010100:Oxygen / Q000378:metabolism*; D010108:Oxyhemoglobins / Q000378:metabolism; D010455:Peptides / Q000032:analysis; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017421:Sequence Analysis
D002712:Chlorides; D006454:Hemoglobins; D010108:Oxyhemoglobins; D010455:Peptides; D011994:Recombinant Proteins; D001216:Asparagine; D000409:Alanine; D010100:Oxygen