Studies of Hemoglobin Denaturation and Heinz Body Formation in the Unstable Hemoglobins

Abstract

A B S T R A C T The sequential changes that occur dur

Insights into Hemoglobin Assembly through in Vivo Mutagenesis of α-Hemoglobin Stabilizing Protein

“…After manual inspection for sequence quality, 31 sequences were trimmed to include only helices 1 and 2 and aligned using the ClustalW algorithm with Strap multiple sequence alignment software (52). 30 Mutations Alter ␣ Chain Binding, Autooxidation, and Hemin Loss-The evolutionarily conserved proline at position 30 in loop 1 of AHSP contacts helix G of ␣-globin at the edge of the protein-binding interface (Fig. 1).…”

“…1). To examine the function of this amino acid, Gell et al (18) made a series of mutations that convert AHSP Pro 30 to Gly, Ala, Val, Phe, and Trp and screened the resultant protein interactions with ␣-globin. Except for the P30G mutant, all AHSP variants showed enhanced affinity (lower K D values) for ferrous ␣CO or ␣O 2 as measured by isothermal titration calorimetry.…”

“…Autooxidation is followed immediately by internal coordination of His 58 (E7) to the hemin iron, resulting in a hexacoordinate bishistidyl or hemichrome structure (18,21,22,28). Bishistidyl hemoglobins occur as ␣-globin degradation products in ␤-thalassemia (4,6,29,30), during normal degradation of hemoglobin, and in some naturally occurring globins in mammalian tissues, plants, certain bacteria, and cold water fish (31)(32)(33)(34)(35)(36)(37)(38)(39)(40). Binding of His 58 to hemin iron appears to inhibit the ability of met-␣ to catalyze ROS production (39,41).…”

“…In the structure of the ASHP⅐met-␣ complex, the cis conformation at the AHSP Asp 29 -Pro 30 peptide bond projects loop 1 toward ␣-globin helix G where AHSP Pro 30 promotes backbone hydrogen bonding interactions. Pro 30 amino acid substitutions in AHSP convert the 29-30 peptide bond to a completely trans conformation, which generally enhances the affinity of AHSP for ferrous ␣CO and ␣O 2 and reduces the rates of autooxidation and subsequent conversion to the bishistidyl form (18).…”

“…Our combined in vitro and in vivo findings refine and revise current models for AHSP function during erythropoiesis and emphasize the physiological importance of high affinity met-␣ binding to AHSP. 30 VariantsDetailed descriptions of measurements of ␣ subunit binding to and release from AHSP variants are given in the accompanying paper by Mollan et al (42). Time courses for association of reduced and oxidized ␣ subunits with AHSP were measured in an Applied Photophysics PiStar kinetic stopped-flow spectrophotometer (Leatherhead, Surrey, UK), monitoring the intrinsic fluorescence of AHSP.…”