N-terminal acetylation and protonation of individual hemoglobin subunits: position-dependent effects on tetramer strength and cooperativity

Abstract

The presence of alanine (Ala) or acetyl serine (AcSer) instead of the normal Val residues at the N-terminals of either the alpha- or the beta-subunits of human adult hemoglobin confers some novel and unexpected features on the protein. Mass spectrometric analysis confirmed that these substitutions were correct and that they were the only ones. Circular dichroism studies indicated no global protein conformational changes, and isoelectric focusing showed the absence of impurities. The presence of Ala at the N-terminals of the alpha-subunits of liganded hemoglobin results in a significantly increased basicity (increased pK(a) values) and a reduction in the strength of subunit interactions at the allosteric tetramer-dimer interface. Cooperativity in O(2) binding is also decreased. Substitution of Ala at the N-terminals of the beta-subunits gives neither of these effects. The substitution of Ser at the N terminus of either subunit leads to its complete acetylation (during expression) and a large decrease in the strength of the tetramer-dimer allosteric interface. When either Ala or AcSer is present at the N terminus of the alpha-subunit, the slope of the plot of the tetramer-dimer association/dissociation constant as a function of pH is decreased by 60%. It is suggested that since the network of interactions involving the N and C termini of the alpha-subunits is less extensive than that of the beta-subunits in liganded human hemoglobin disruptions there are likely to have a profound effect on hemoglobin function such as the increased basicity, the effects on tetramer strength, and on cooperativity.

Figure 1.

Mass spectrometry analysis. (A) ESI/MS analysis of hemoglobin sample α₂^AcSβ₂. The ion envelopes for α and β chains are shown in the raw m/z spectrum. (Inset) Transformed mass spectrum after deconvolution of the ion envelopes encompassing the region of charge states 11+ through 20+ (m/z range 750–1500). (Refer to text for experimental details.) (B) MALDI-QqTOF/MS spectrum of a tryptic digest of hemoglobin sample α₂^AcSβ₂. Peaks are labeled as α- or β-chain peptides followed by T for trypsin and the number of the first and last residues in the peptide in accordance with the numbering system for the primary structure of the entire polypeptide chain. The combined MALDI data yielded information covering 100% of both α and β globin chains. (C) ESI/MS/MS analysis of the singly charged peak of the α-chain N-terminal tryptic peptide αT(1–7) from hemoglobin sample α₂^AcSβ₂. The underlined residue was mutated. Only b and y ions are labeled for clarity. Unidentified peaks are labeled with an asterisk. The mutated residue was assigned based on the occurrence of ions y₆⁺ and y₇⁺ and the PTC-derivative of the same peptide. (Refer to text for experimental details.) (D) ESI/MS/MS hypothesis-driven analysis of the N-terminal tryptic peptide αT(1–7) of human hemoglobin (row a) and recombinant hemoglobin samples α₂^Aβ₂ (row b), α₂β₂^A (row c) and α₂^Aβ₂^A (row d). Spectra show the isolated precursor ions selected based on either the presence or absence of the Val → Ala modification. The hypothetical precursor ions were subsequently fragmented.

Figure 1.

Mass spectrometry analysis. (A) ESI/MS analysis of hemoglobin sample α₂^AcSβ₂. The ion envelopes for α and β chains are shown in the raw m/z spectrum. (Inset) Transformed mass spectrum after deconvolution of the ion envelopes encompassing the region of charge states 11+ through 20+ (m/z range 750–1500). (Refer to text for experimental details.) (B) MALDI-QqTOF/MS spectrum of a tryptic digest of hemoglobin sample α₂^AcSβ₂. Peaks are labeled as α- or β-chain peptides followed by T for trypsin and the number of the first and last residues in the peptide in accordance with the numbering system for the primary structure of the entire polypeptide chain. The combined MALDI data yielded information covering 100% of both α and β globin chains. (C) ESI/MS/MS analysis of the singly charged peak of the α-chain N-terminal tryptic peptide αT(1–7) from hemoglobin sample α₂^AcSβ₂. The underlined residue was mutated. Only b and y ions are labeled for clarity. Unidentified peaks are labeled with an asterisk. The mutated residue was assigned based on the occurrence of ions y₆⁺ and y₇⁺ and the PTC-derivative of the same peptide. (Refer to text for experimental details.) (D) ESI/MS/MS hypothesis-driven analysis of the N-terminal tryptic peptide αT(1–7) of human hemoglobin (row a) and recombinant hemoglobin samples α₂^Aβ₂ (row b), α₂β₂^A (row c) and α₂^Aβ₂^A (row d). Spectra show the isolated precursor ions selected based on either the presence or absence of the Val → Ala modification. The hypothetical precursor ions were subsequently fragmented.

Figure 2.

Isoelectric focusing of purified recombinant Hb mutants. Approximately 5 μg of each Hb was applied to a pH 6.0–8.0 range gel (Hb Resolve, Perkin-Elmer Life Sciences). Electrophoresis was performed for 30 min at 600 V and then for 45 min at 900 V at 10°C. The gel was stained with the JBZ stain (Perkin-Elmer-Wallach). The anode is at the top and the cathode is at the bottom. The standard hemoglobins on the far left and the far right are hemoglobins A, F, S, and C. The identity of each recombinant hemoglobin is shown under each lane. A small superscript “A” indicates an N-terminal Ala on that subunit. A small superscript “AcS” indicates an N-terminal acetylserine on that subunit. Subunits without superscripts contain N-terminal Val, e.g., α₂β₂ is HbA.

Figure 3.

Circular dichroism spectra were recorded as described in the text. An Hb concentration of 10 μM as the tetramer was used in the far-ultraviolet region. (A) Samples α₂β₂, α₂β₂^A, and α₂β₂^AcS; (B) samples α₂β₂ and α₂^AcSβ₂; (C) samples α₂β₂ and α₂^Aβ₂.

Figure 4.

Tetramer–dimer dissociation constants at pH 7.5. The gel filtration procedure used to determine these tetramer–dimer dissociation constants (K_d values) and the treatment of the data have been described in detail in Manning et al. (1996, . The plots show the amounts of Hb present on the column during the analysis to attain a certain percent of tetramer, the functional O₂-carrying structure of Hb. Further treatment of the data is shown in the inset where the K_d value can be read directly as the intersection point of the horizontal line at 1 on the Y axis with the experimental line.

Figure 5.

Association constants for tetramer–dimer assembly as a function of pH. Dissociation constants (K_d) measured as in Figure 4 ▶, were determined at each pH indicated and then converted to association constants (K_a=¹/K_d). The data for α₂γ₂ (HbF) (dashed line), α₂β₂ (HbA), and α₂γ₂^AcG (HbF₁) have been reported previously (Manning and Manning 2001). The symbols for each sample are α₂γ₂, ▴; α₂β₂, ▵; α₂γ₂^AcG, x; α₂β₂^A,•; α₂β₂^AcS, ▪; α₂^AcSβ₂, □, α₂^Aβ₂, ○.

Figure 6.

Hill Plots for N-terminal substituted mutant hemoglobins. The samples are identified on each panel: (upper left) α₂^Aβ₂, (upper right) α₂^AcSβ₂, (lower left) α₂β₂^A, (lower right) α₂β₂^AcS. These Hill plots were calculated from the O₂-binding curve in the presence of 2,3-DPG. The points on each line were read from the HemOScan chart paper at each 5% O₂-tension between 20% and 70% O₂ saturation. The best fit line shown was then drawn through these points with weighting on the mid-range values. These data are from the O₂-binding data in the presence of 2,3-DPG at pH 7.5. A similar lowering of the Hill coefficient for the N-terminal α-subunit substitutions was found for the O₂-binding data for Hb alone and for Hb with chloride.

Figure 7.

N-terminal/C-terminal interactions for liganded human hemoglobin A. (A) α–α interactions; (B) β–β interactions. The coordinates used are from PDB=5 HHO by Shaanan (1983). The α-subunits in α–α are designated as chains A and C and the β-subunits in β–β are designated as chains B and D. In α–α the white lines with numbers (in angstroms) are H-bonds. The 2.24 Å distance is from one carboxylate oxygen of Arg-141 to the α-NH₂ of Val-1. The 2.92 Å distance is from the same carboxylate oxygen of Arg-141 to the peptide bond N between Val-1 and Leu-2. The 2.40 Å distance is between the other carboxylate oxygen of Arg-141 to the ɛ-NH₂ of Lys-127. In β–β the white lines from Val-98 are H-bonds between the peptide bond N and O of Val-98 to the phenolic OH of Tyr-145, whereas those between Val-1, His-2, Lys-144, and His-146 are distances. The 3.09 Å distance is between one carboxylate oxygen of His-146 and the ɛ-NH₂ of Lys-144. The 5.10 Å distance is between the other carboxylate oxygen of His-146 and the α-NH₂ of Val-1. The 6.08 Å distance is between the ɛ-NH₂ of Lys-144 and the peptide bond O between Val-1 and His-2. The program Insight II (Accelrys) was used to construct these figures from the data of Shaanan (1983).