ABSTRACT
When first tested for abnormal hemoglobins, a 2-year-old boy, appeared to have Hb F, Hb S and Hb A2. Confirmatory testing revealed a beta chain variant inherited from his father and beta S from his mother. Analysis of tryptic peptides in conjunction with automated DNA sequence analysis showed that the variant hemoglobin was Hb Shelby [beta 131(H9)Gln-->Lys (CAG-->AAG)]. Heat and mechanical stabilities of various liganded Hb Shelby tetramers were compared to those of Hb A and Hb S. Oxy-Hb Shelby precipitated more readily than oxy-Hb A, but was much more stable than oxy-Hb S during mechanical agitation. In contrast, oxy-Hb Shelby was much less stable than oxy-Hb A and oxy-Hb S following heat treatment. Met-Hb Shelby was most unstable compared to other liganded forms of Hb Shelby, while deoxy- and carbonmonoxy-forms of Hb Shelby showed similar heat-induced precipitation rates. These data indicate that heat instability of Hb Shelby is accompanied by heme oxidation, and that denaturation by mechanical agitation occurs in the absence of heme oxidation. Hb Shelby, like Hb A, can form hybrids with Hb S which participate in polymer formation in vitro. However, Hb S/Hb Shelby hybrids copolymerized with Hb S less than A/S hybrids. Since the patient's MCHC value is normal, this finding coupled with the elevated Hb A2 and Hb F levels, both of which are known to inhibit polymerization of Hb S, may contribute to the patient's mild clinical presentation.
Subject(s)
Globins/genetics , Hemoglobins, Abnormal/genetics , Sickle Cell Trait/genetics , Base Sequence , Child, Preschool , Female , Hemoglobin, Sickle/chemistry , Heterozygote , Hot Temperature , Humans , Ligands , Male , Molecular Sequence Data , Polymers , Protein Denaturation , Solubility , Stress, MechanicalABSTRACT
In an effort to clarify the role of amino acid hydrophobicity at the beta 6 position in sickling we have made recombinant hemoglobin tetramers containing beta 6 Val (Hb S) and beta 6 Leu (Hb Leu). Recombinant Hb S and Hb Leu had the same electrophoretic mobility, chromatographic behavior, and absorption spectrum. The deoxy form of both tetramers polymerized in high phosphate buffer (1.8 M) and exhibited distinct delay times prior to polymerization. The kinetics of polymerization for recombinant and native Hb S were similar, while recombinant Hb Leu polymerized more readily. The solubility of deoxy Hb Leu was less than deoxy Hb S, indicating that rapid polymerization and decreased solubility of deoxyhemoglobin is accelerated with increasing hydrophobicity at the beta 6 position.
Subject(s)
Hemoglobin, Sickle/chemistry , Hemoglobins, Abnormal/chemistry , Recombinant Fusion Proteins/chemistry , Amino Acid Sequence , Hemoglobin, Sickle/genetics , Hemoglobins/chemistry , Hemoglobins, Abnormal/genetics , Humans , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Polymers , SolubilityABSTRACT
The biochemical, spectroscopic, enzymatic, and redox properties of spleen myeloperoxidase, a peroxidase formerly called "spleen green hemeprotein," and granulocyte myeloperoxidase were compared by electrophoresis, amino-terminal amino acid sequences, optical and EPR spectroscopy, steady-state enzyme kinetics, and oxidation-reduction potential. The spleen enzyme exhibits properties indistinguishable from those of the granulocyte enzyme. We conclude that the spleen enzyme is indeed identical to granulocyte myeloperoxidase.