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Application of shielding boronate affinity chromatography in the study of the glycation pattern of haemoglobin

Li, YuCai; Jeppsson, Jan-Olof LU ; Jörnten-Karlsson, Magnus; Linné Larsson, Eva; Jungvid, Hans; Galaev, Igor LU and Mattiasson, Bo LU (2002) In Journal of Chromatography. B 776(2). p.149-160
Abstract
Human haemoglobin (Hb) may appear in a number of glycated species. The glycation pattern of Hb using shielding boronate affinity chromatography (SBAC) has been studied in the present work. SBAC is a novel separation technique, which eliminates nonspecific boronate-protein interactions by introducing a so-called shielding reagent [1,2]. Two samples from Bio-Rad (Lyphochek(R))-one from normal persons' blood with relatively low HbA(1c) level (HbL) and the other from diabetic patients' blood with an elevated HbA(1c) level (HbH)-were used for the investigation. Glycated Hb (GHb) was separated from nonglycated Hb species using Tris as the shielding reagent. Two eluted peaks, eluted peak 1 (E1) and eluted peak 2 (E2), were obtained using a linear... (More)
Human haemoglobin (Hb) may appear in a number of glycated species. The glycation pattern of Hb using shielding boronate affinity chromatography (SBAC) has been studied in the present work. SBAC is a novel separation technique, which eliminates nonspecific boronate-protein interactions by introducing a so-called shielding reagent [1,2]. Two samples from Bio-Rad (Lyphochek(R))-one from normal persons' blood with relatively low HbA(1c) level (HbL) and the other from diabetic patients' blood with an elevated HbA(1c) level (HbH)-were used for the investigation. Glycated Hb (GHb) was separated from nonglycated Hb species using Tris as the shielding reagent. Two eluted peaks, eluted peak 1 (E1) and eluted peak 2 (E2), were obtained using a linear gradient elution with Tris. Several bands were observed on isoelectric focusing gel, which showed the same migration positions as Hb adducts, such as HbA(o), which is major Hb component containing two alpha chains and two beta chains; HbA(1c) which is post-translational glycation on the N-terminus of the beta chains of HbA(o); Foetal Hb (HbF), consisting of two alpha chains and two gamma chains; and glutathione Hb (also called HbSSG), which is the result from thiol-disulphide interchain exchange during oxidation of the thiol groups of Hb. In both HbL and HbH samples, E2 exhibited slightly higher amounts of HbF than E1. Electrospray-ionisation mass spectrometry showed that: (1) HbL-E1 was glycated with single glucose on both alpha and beta chains while no observable glycated chains were present in HbL-E2; (2) both HbH-E1 and HbH-E2 were glycated with single glucoses on both alpha and beta chains, however, compared with HbH-E1, HbH-E2 showed a higher relative intensity of the glycated beta chain and lower relative intensity of the glycated alpha chain; and (3) the degree of glycation increased with increasing glycation level of the sample. The amount of HbA(1c) presented in the eluted peaks was further determined using enzymatic digestion of glycated Hb by endoproteinase Glu-C and the subsequent separation and analysis of the digested peptides by reversed-phase high-performance liquid chromatography and capillary electrophoresis. The values of HbA(1c)/HbA(o) of the eluted peaks, i.e. HbL-E1, HbL-E2, HbH-E1 and HbH-E2, were 0.27, 0.19, 0.50 and 0.43, respectively. In both HbL and HbH samples, E1 contained higher amounts of HbA(1c) than E2. This study demonstrates the structural heterogeneity of GHb as well as the possibility of using SBAC to detect glycated species of Hb. (Less)
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publishing date
type
Contribution to journal
publication status
published
subject
keywords
shielding boronate affinity chromatography, glycation pattern, haemoglobin
in
Journal of Chromatography. B
volume
776
issue
2
pages
149 - 160
publisher
Elsevier
external identifiers
  • wos:000177451900002
  • pmid:12137996
  • scopus:0037026426
ISSN
1873-376X
DOI
10.1016/S1570-0232(02)00162-9
language
English
LU publication?
yes
id
bb710f39-8f2b-4874-b231-8896360e8cae (old id 329179)
date added to LUP
2007-11-14 13:55:22
date last changed
2017-08-20 03:29:58
@article{bb710f39-8f2b-4874-b231-8896360e8cae,
  abstract     = {Human haemoglobin (Hb) may appear in a number of glycated species. The glycation pattern of Hb using shielding boronate affinity chromatography (SBAC) has been studied in the present work. SBAC is a novel separation technique, which eliminates nonspecific boronate-protein interactions by introducing a so-called shielding reagent [1,2]. Two samples from Bio-Rad (Lyphochek(R))-one from normal persons' blood with relatively low HbA(1c) level (HbL) and the other from diabetic patients' blood with an elevated HbA(1c) level (HbH)-were used for the investigation. Glycated Hb (GHb) was separated from nonglycated Hb species using Tris as the shielding reagent. Two eluted peaks, eluted peak 1 (E1) and eluted peak 2 (E2), were obtained using a linear gradient elution with Tris. Several bands were observed on isoelectric focusing gel, which showed the same migration positions as Hb adducts, such as HbA(o), which is major Hb component containing two alpha chains and two beta chains; HbA(1c) which is post-translational glycation on the N-terminus of the beta chains of HbA(o); Foetal Hb (HbF), consisting of two alpha chains and two gamma chains; and glutathione Hb (also called HbSSG), which is the result from thiol-disulphide interchain exchange during oxidation of the thiol groups of Hb. In both HbL and HbH samples, E2 exhibited slightly higher amounts of HbF than E1. Electrospray-ionisation mass spectrometry showed that: (1) HbL-E1 was glycated with single glucose on both alpha and beta chains while no observable glycated chains were present in HbL-E2; (2) both HbH-E1 and HbH-E2 were glycated with single glucoses on both alpha and beta chains, however, compared with HbH-E1, HbH-E2 showed a higher relative intensity of the glycated beta chain and lower relative intensity of the glycated alpha chain; and (3) the degree of glycation increased with increasing glycation level of the sample. The amount of HbA(1c) presented in the eluted peaks was further determined using enzymatic digestion of glycated Hb by endoproteinase Glu-C and the subsequent separation and analysis of the digested peptides by reversed-phase high-performance liquid chromatography and capillary electrophoresis. The values of HbA(1c)/HbA(o) of the eluted peaks, i.e. HbL-E1, HbL-E2, HbH-E1 and HbH-E2, were 0.27, 0.19, 0.50 and 0.43, respectively. In both HbL and HbH samples, E1 contained higher amounts of HbA(1c) than E2. This study demonstrates the structural heterogeneity of GHb as well as the possibility of using SBAC to detect glycated species of Hb.},
  author       = {Li, YuCai and Jeppsson, Jan-Olof and Jörnten-Karlsson, Magnus and Linné Larsson, Eva and Jungvid, Hans and Galaev, Igor and Mattiasson, Bo},
  issn         = {1873-376X},
  keyword      = {shielding boronate affinity chromatography,glycation pattern,haemoglobin},
  language     = {eng},
  number       = {2},
  pages        = {149--160},
  publisher    = {Elsevier},
  series       = {Journal of Chromatography. B},
  title        = {Application of shielding boronate affinity chromatography in the study of the glycation pattern of haemoglobin},
  url          = {http://dx.doi.org/10.1016/S1570-0232(02)00162-9},
  volume       = {776},
  year         = {2002},
}