Properties of a recombinant human hemoglobin double mutant: sickle hemoglobin with Leu-88(beta) at the primary aggregation site substituted by Ala.
A recombinant double mutant of hemoglobin (Hb), E6V/L88A(beta), was constructed to study the strength of the primary hydrophobic interaction in the gelation of sickle Hb, i.e., that between the mutant Val-6(beta) of one tetramer and the hydrophobic region between Phe-85(beta) and Leu-88(beta) on an adjacent tetramer. Thus, a construct encoding the donor Val-6(beta) of the expressed recombinant HbS and a second mutation encoding an Ala in place of Leu-88(beta) was assembled. The doubly mutated beta-globin gene was expressed in yeast together with the normal human alpha-chain, which is on the same plasmid, to produce a soluble Hb tetramer. Characterizations of the Hb double mutant by mass spectrometry, by HPLC, and by peptide mapping of tryptic digests of the mutant beta-chain were consistent with the desired mutations. The absorption spectra in the visible and the ultraviolet regions were practically superimposable for the recombinant Hb and the natural Hb purified from human red cells. Circular dichroism studies on the overall structure of the recombinant Hb double mutant and the recombinant single mutant, HbS, showed that both were correctly folded. Functional studies on the recombinant double mutant indicated that it was fully cooperative. However, its gelation concentration was significantly higher than that of either recombinant or natural sickle Hb, indicating that the strength of the interaction in this important donor-acceptor region in sickle Hb was considerably reduced even with such a conservative hydrophobic mutation.
Martin de Llano
J J
JJ
Rockefeller University, New York, New York 10021.
Manning
J M
JM
D000409:Alanine; D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002942:Circular Dichroism; D005782:Gels; D005914:Globins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006451:Hemoglobin, Sickle / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007525:Isoelectric Focusing; D007930:Leucine; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013053:Spectrophotometry; D014357:Trypsin / Q000378:metabolism
D005782:Gels; D006451:Hemoglobin, Sickle; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D005914:Globins; D014357:Trypsin; D007930:Leucine; D000409:Alanine; D010100:Oxygen