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

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 ®)—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 two 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 relative intensity of the glycated 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.