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

摘要

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)-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_0, which is major Hb component containing two α chains and tow β chains; HbA_(1c), which is post-translational glycation on the N-terminus of the β chains of HbA_0; Foetal Hb (HbF), consisting of two α chains and two γ 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 α and β 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 α and β chains, however, compared with HbH-E1, HbH-E2 showed a higher reliative intensity of the lgycated β chain and lower relative intensity of the glycated α 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_0 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.