Structural analysis of glycated human hemoglobin using native mass spectrometry (original) (raw)
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Journal of biomolecular techniques : JBT, 2011
MALDI-TOF mass spectrometry is used here to differentiate different glycoisoforms of normal and variant hemoglobins (Hbs) in nonenzymatic in vitro glycation. Single, double, and/or multiple glycation of the α-globin, β-globin, and/or γ-globin is observed. Different glycation rates are observed for various Hbs, and the normal Hb A has the slowest rate. Although the Hb A is relatively stable upon condensation with glucose at 37°C, the variants Hb C, Hb E, Hb F, Hb Leiden, and Hb San Diego are less stable. In addition, data reveal that the number of glucose attached/Hb molecule (state of glycation) increases with longer incubation time, higher glucose concentration, and higher temperature. The pH dependence of the state of glycation is more complex and varies for different Hbs. Although pH has little effect on the state of glycation for Hb C, Hb E, and Hb Leiden, it increases for Hb A and Hb F upon changing the pH of the solution from phosphate buffer saline (pH 7.4) to carbonate buffe...
MALDI-TOF MS is used successfully in investigating in vitro glycation of normal and variant hemoglobins (Hbs). Singly glycated, doubly glycated, and/or multiply glycated glycoisoforms of the alpha-globin, beta-globin, and gamma-globin of Hbs are observed. Different glycation rates are observed for normal and variant Hbs, with the normal Hb A having the slowest rate. The normal Hb A is more stable than the Hb C, Hb E, Hb F, Hb Leiden, and Hb San Diego upon condensation with glucose at 37°C. Data reveal that with longer incubation time (up to 5 d), higher glucose concentration (up to 1 M), and higher temperature (up to 37°C), the number of glycated amino acid residues of Hbs increase. The extent of the glycation of both Hb A and Hb F increases upon changing the solvent from PBS (pH 7.4) to carbonate buffer (pH 10). However, this pH change has a lesser effect on the glycation of the Hb C, Hb E, or Hb Leiden. In this study, higher concentration of the glucose is used to increase the rea...
2000
Hemoglobin A1c (HbA1c) is a stable minor Hb variant formed in vivo by posttranslational modification by glucose, originally identified by using cation exchange chromatography, and containing primarily glycated N- terminal b-chains. However, the structure(s) of the quantified species has not been elucidated, and the available methods lack a reference standard. We used electrospray ionization mass spectrometry to determine the extent
Hemoglobin, 2019
This is the first report of quadrupole time-of-flight (Q-TOF) mass spectrometric identification of the hemoglobin (Hb) subunits, a, b, d and c peptides, derived from enzymatic-digestion of proteins in the early unknown peaks of the cation exchange chromatography of Hb. The objectives were to identify the unknown high performance liquid chromatography (HPLC) peaks in healthy subjects and in patients with b-thalassemia (b-thal). The results demonstrate the existence of pools of free globin chains in red blood cells (RBCs). The a-, b-, dand c-globin peptides were identified in the unknown HPLC peaks. The quantification and role of the free globin pool in patients with b-thal requires further investigation. Identification of all types of Hb subunits in the retention time (RT) before 1 min. suggests that altered Hbs is the nature of these fast-eluting peaks. Relevancy of thalassemias to the proteinaggregation disorders will require review of the role of free globin in the pathology of the disease.
Functional and subunit assembly properties of hemoglobin alberta (α2β2101 Glu → Gly)
Journal of Molecular Biology, 1985
Hemoglobin Alberta has an amino acid substitution at position 101 (Glu + Gly), a residue involved in the xl/?2 contact region of both the deoxy and oxy conformers of normal adult hemoglobin. Oxygen equilibrium measurements of stripped hemoglobin Alberta at 20°C in the absence of phosphate revealed a high affinity (P,, = 0.75 mm Hg at pH 7), cooperative hemoglobin variant (n = 2.3 at pH 7) with a normal Bohr effect (-A log P,,/ApH(,-s, = O-65). The addition of inositol hexaphosphate resulted in a decrease in oxygen affinity (P,, = 8.2 mm Hg at pH 7), a slight increase in the value of n and an enhanced Bohr effect. Rapid mixing experiments reflected the equilibrium results. A rapid rate of carbon monoxide binding (I' = 7-O x lo5 M-l s-l) and a slow rate of overall oxygen dissociation (k = 15 s-l) was seen at pH 7 and 20°C in the absence of phosphate. Under these experimental conditions the tetramer stability of liganded and unliganded hemoglobin Alberta was investigated by spectrophotometric kinetic techniques. The 4K4 value (the liganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta was found to be 0.83 x 10e6 M compared to a 4K4 value for hemoglobin A of 2.3 x 10m6 M, indicating that the Alberta tetramer was less dissociated into dimers than the tetramer of hemoglobin A. The values of OK, (the unliganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta and hemoglobin A were also measured and found to be 2.5 x lo-* M and 1.5 x 10-l' M, respectively, demonstrating a greatly destabilized deoxyhemoglobin tetramer for hemoglobin Alberta compared to deoxyhemoglobin A. The functional and subunit dissociation properties of hemoglobin Alberta appear to be directly related to the dual role of the /3101 residue in stabilizing the tetrameric form of the liganded structure, while concurrently destabilizing the unliganded tetramer molecule.
Bioconjugate Chemistry, 2011
Glutathionyl hemoglobin, an example of post-translationally modified hemoglobin, has been studied as a marker of oxidative stress in various diseased conditions. Compared to normal hemoglobin, glutathionyl hemoglobin has been found to have increased oxygen affinity and reduced cooperativity. However, detailed information concerning the structural perturbation of hemoglobin associated with glutathionylation is lacking. In the present study, we report structural changes associated with glutathionylation of deoxyhemoglobin by hydrogen/deuterium (H/D) exchange coupled to matrix assisted laser desorption ionization (MALDI) mass spectrometry. We analyzed isotope exchange kinetics of backbone amide hydrogen of eleven peptic peptides in the deoxy state of both hemoglobin and glutathionyl hemoglobin molecules. Analysis of the deuterium incorporation kinetics for both molecules showed structural changes associated with the following peptides: R34-46, R1-29, β32-41, β86-102, β115-129, and β130-146. H/D exchange experiments suggest that glutathionylation of hemoglobin results in a change in conformation located at the above-mentioned regions of the hemoglobin molecule. MALDI mass spectrometry based H/D exchange experiment might be a simple way of monitoring structural changes associated with post-translational modification of protein.
Glycated Hemoglobin-The Clinical and Biochemical Divide: A Review
2011
Diabetes mellitus is a chronic metabolic disorder characterized by rise in blood glucose level called "hyperglycaemia". The main long term vascular complications are coronary artery disease, stroke, renal failure etc. The measurement of glycosylated hemoglobin (GHb) is one of the well established means of monitoring glycemic control in patients with diabetes mellitus. Hemoglobin (Hb) is composed of four globin chains. Adult hemoglobin (HbA) is the most abundant form in most adults and consists of two α and two β chains. Fetal hemoglobin (HbF), which is predominantly present at birth, consists of two α and two γ chains. Glycosylation is a non- enzymatic reaction between free aldehyde group of glucose and free amino groups of proteins. The biosynthesis of glycosylated hemoglobins (HbA1a, HbA1b, and HbA1c) occurs slowly, continuously and almost irreversibly throughout the four month life span of erythrocytes and the process is non-enzymatic. Recent reports have shown that the...