Properties of a recombinant human hemoglobin with aspartic acid 99(β), an important intersubunit contact site, substituted by lysine

H. Yanase, Sean M. Cahill, J. J M De Llano, L. R. Manning, K. Schneider, B. T. Chait, K. D. Vandegriff, R. M. Winslow, J. M. Manning

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Site-directed mutagenesis of an important subunit contact site, Asp- 99(β), by a Lys residue (D99K(β)) was proven by sequencing the entire β- globin gene and the mutant tryptic peptide. Oxygen equilibrium curves of the mutant hemoglobin (Hb) (2-15 mM in heme) indicated that it had an increased oxygen affinity and a lowered but significant amount of cooperativity compared to native HbA. However, in contrast to normal HbA, oxygen binding of the recombinant mutant Hb was only marginally affected by the allosteric regulators 2,3-diphosphoglycerate or inositol hexaphosphate and was not at all responsive to chloride. The efficiency of oxygen binding by HbA in the presence of allosteric regulators was limited by the mutant Hb. At concentrations of 0.2 mM or lower in heme, the mutant D99K(β) Hb was predominantly a dimer as demonstrated by gel filtration, haptoglobin binding, fluorescence quenching, and light scattering. The purified dimeric recombinant Hb mutant exists in 2 forms that are separable on isoelectric focusing by about 0.1 pH unit, in contrast to tetrameric hemoglobin, which shows 1 band. These mutant forms, which were present in a ratio of 60:40, had the same masses for their heme and globin moieties as determined by mass spectrometry. The elution positions of the α- and β-globin subunits on HPLC were identical. Circular dichroism studies showed that one form of the mutant Hb had a negative ellipticity at 410 nm and the other had positive ellipticity at this wavelength. The findings suggest that the 2 D99K(β) recombinant mutant forms have differences in their heme-protein environments.

Original languageEnglish (US)
Pages (from-to)1213-1223
Number of pages11
JournalProtein Science
Volume3
Issue number8
StatePublished - 1994
Externally publishedYes

Fingerprint

Aspartic Acid
Lysine
Hemoglobins
Globins
Heme
Oxygen
Hemeproteins
2,3-Diphosphoglycerate
Mutagenesis
Phytic Acid
Haptoglobins
Isoelectric Focusing
Circular Dichroism
Site-Directed Mutagenesis
Dimers
Light scattering
Gel Chromatography
Mass spectrometry
Chlorides
Quenching

Keywords

  • cooperativity
  • hemoglobin
  • hemoglobin intersubunit contact
  • mass spectrometry
  • mutagenesis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Yanase, H., Cahill, S. M., De Llano, J. J. M., Manning, L. R., Schneider, K., Chait, B. T., ... Manning, J. M. (1994). Properties of a recombinant human hemoglobin with aspartic acid 99(β), an important intersubunit contact site, substituted by lysine. Protein Science, 3(8), 1213-1223.

Properties of a recombinant human hemoglobin with aspartic acid 99(β), an important intersubunit contact site, substituted by lysine. / Yanase, H.; Cahill, Sean M.; De Llano, J. J M; Manning, L. R.; Schneider, K.; Chait, B. T.; Vandegriff, K. D.; Winslow, R. M.; Manning, J. M.

In: Protein Science, Vol. 3, No. 8, 1994, p. 1213-1223.

Research output: Contribution to journalArticle

Yanase, H, Cahill, SM, De Llano, JJM, Manning, LR, Schneider, K, Chait, BT, Vandegriff, KD, Winslow, RM & Manning, JM 1994, 'Properties of a recombinant human hemoglobin with aspartic acid 99(β), an important intersubunit contact site, substituted by lysine', Protein Science, vol. 3, no. 8, pp. 1213-1223.
Yanase, H. ; Cahill, Sean M. ; De Llano, J. J M ; Manning, L. R. ; Schneider, K. ; Chait, B. T. ; Vandegriff, K. D. ; Winslow, R. M. ; Manning, J. M. / Properties of a recombinant human hemoglobin with aspartic acid 99(β), an important intersubunit contact site, substituted by lysine. In: Protein Science. 1994 ; Vol. 3, No. 8. pp. 1213-1223.
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abstract = "Site-directed mutagenesis of an important subunit contact site, Asp- 99(β), by a Lys residue (D99K(β)) was proven by sequencing the entire β- globin gene and the mutant tryptic peptide. Oxygen equilibrium curves of the mutant hemoglobin (Hb) (2-15 mM in heme) indicated that it had an increased oxygen affinity and a lowered but significant amount of cooperativity compared to native HbA. However, in contrast to normal HbA, oxygen binding of the recombinant mutant Hb was only marginally affected by the allosteric regulators 2,3-diphosphoglycerate or inositol hexaphosphate and was not at all responsive to chloride. The efficiency of oxygen binding by HbA in the presence of allosteric regulators was limited by the mutant Hb. At concentrations of 0.2 mM or lower in heme, the mutant D99K(β) Hb was predominantly a dimer as demonstrated by gel filtration, haptoglobin binding, fluorescence quenching, and light scattering. The purified dimeric recombinant Hb mutant exists in 2 forms that are separable on isoelectric focusing by about 0.1 pH unit, in contrast to tetrameric hemoglobin, which shows 1 band. These mutant forms, which were present in a ratio of 60:40, had the same masses for their heme and globin moieties as determined by mass spectrometry. The elution positions of the α- and β-globin subunits on HPLC were identical. Circular dichroism studies showed that one form of the mutant Hb had a negative ellipticity at 410 nm and the other had positive ellipticity at this wavelength. The findings suggest that the 2 D99K(β) recombinant mutant forms have differences in their heme-protein environments.",
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AU - Manning, L. R.

AU - Schneider, K.

AU - Chait, B. T.

AU - Vandegriff, K. D.

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