Analyses of in vitro nonenzymatic glycation of normal and variant hemoglobins by MALDI-TOF mass spectrometry (original) (raw)
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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...
Structural analysis of glycated human hemoglobin using native mass spectrometry
The FEBS Journal
Glycated hemoglobin (GHb) is the indicator of the long-term glycemic index of an individual. GHb is formed by the irreversible modification of N-terminal a-amino group of b globin chain with glucose via Amadori rearrangement. Cation exchange chromatography exploits the difference in surface charges between GHb and native hemoglobin (HbA 0) for their separation and quantification. However, glucose condensation is specific to primary amino groups. Therefore, structural characterization of GHb synthesized in vivo is essential as multiple glycation may interfere with GHb assessment. The stoichiometric composition of different glycated hemoglobin from a 19% GHb sample was deduced using native mass spectrometry. We observed a comparable population of a and b glycated tetramers for mono-glycated HbA 0. Surprisingly, doubly and triply glycated HbA 0 also showed mono-glycated a and b globins. Thus, we propose that glycation of hemoglobin (HbA) occurs symmetrically across a and b globins with preference to unmodified globin first. Correlation between conventional and mass spectrometry-based quantification of GHb showed a reliable estimation of the glycemic index of individuals carrying HbA 0. Mutant HbAs have different retention time than HbA 0 due to the differences in their surface charge. Thus, their glycated analog may elute at different retention time compared to GHb. Consequently, our method would be ideal for assessing the glycemic index of an individual carrying mutant HbA.
The relative extent of glycation of haemoglobin and albumin
Clinica Chimica Acta, 1987
The level of non-enzymatic glycation of a protein is thought to depend on the number of sites available for reaction, the half-life of the protein and the ambient concentration of glucose. Accordingly, the modification of two blood proteins with a similar number of potential sites but different survival times was examined in non-diabetic patients by periodate oxidation and by reduction with [ 3H]borohydride. The amount of glycation of haemoglobin and its sub-fractions HbA, and HbA,, were determined to be 0.44, 2.42 and 2.24 mol/mol respectively and the corresponding value for albumin was 0.37 mol/mol protein. Amino acid analysis showed that the c amino groups of albumin were more extensively modified than they were in haemoglobin and thus it is concluded that the average rate of reaction of the lysine residues in albumin is markedly faster than in haemoglobin.
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...
Structural alterations of hemoglobin and myoglobin by glyoxal: A comparative study
International Journal of Biological Macromolecules, 2014
Glyoxal, a highly reactive oxoaldehyde, increases in diabetic condition. It reacts with different proteins to form advanced glycation end products (AGEs). Here we have studied the structural alterations as well as the sites and nature of amino acid modifications of two heme proteins, hemoglobin and myoglobin on incubation with glyoxal for seven days at 25 • C. In comparison with normal hemoglobin (HbA 0), glyoxal-treated hemoglobin (GHbA 0) exhibits decreased absorbance around 280 nm, reduced intrinsic fluorescence and lower surface hydrophobicity. However, glyoxal-treated myoglobin (GMb) exhibits the opposite effects in these respects when compared to normal myoglobin (Mb). Glyoxal increases the thermal stability of hemoglobin, while it decreases the stability of myoglobin. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)-mass spectrometry reveals modifications of Arg-31␣, Arg-40 and Arg-104 of hemoglobin by glyoxal to hydroimidazolone adducts. On the other hand, glyoxal modifies Lys-133 and Lys-145 to carboxymethyllysine and Arg-31 to hydroimidazolone adducts in myoglobin. Thus the same oxoaldehyde exerts different effects on hemoglobin and myoglobin and may be associated with different structural properties of the proteins.
Glycosylated haemoglobins and the oxygen affinity of whole blood
Diabetologia, 1982
The pOz at which haemoglobin is half-saturated with oxygen (p50) was determined at fixed pCO2 (45 mmHg) and without altering the resulting pH and the level of organic phosphates in heparinized whole blood samples from 26 diabetic patients and 24 normal subjects of both sexes. Diabetic blood p50 was higher (29.79 + 1.68 versus 28.26 _ 1.16 mmHg, p < 0.001) and with a higher 2,3-diphosphoglyceric acid/haemoglobin molar ratio (1.04 + 0.15 versus 0.86 + 0.10, p < 0.001). The pH at a pCO2 of 45 mmHg was the same in the two groups. The observed p50 values were compared with those obtained after normalization in respect to pH and the level of 2,3-diphosphoglyceric acid. We conclude that glycosylated haemoglobins, known to have an increased affinity for oxygen when purified and in diluted solutions, do not play a significant role in the oxygen affinity pattern of diabetics at the concentrations normally found in vivo.
2005
Allen in 1958 showed chromatographic heterogeneity of hemoglobin A (1). Rahbar in Taheran first demonstrated elevation of minor hemoglobin in diabetes mellitus in 1968 (2). Trivelli introduced the column method of seperating out the fast hemoglobins in 1971 (3). Bunn and co-workers in 1978 pointed out that hemoglobin Alc is related to control of diabetes (4). The column method used in other countries, both the micro and macro columns have been expensive and as we shall discuss later, they are subject to more errors than the chemical method. Therefore, we have been interested in the chemical method that was described by Fluckiger and Winterhalter in 1976 (5). We modified this method to some extent to suit our needs and have tested it out over the past 5 years and found it to be working satisfactorily (6). This method has the advantage of not being influenced by changes in the ionic charge of the hemoglobin which occurs in many hemoglobinopathies and which occurs following aspirin or ...