Separation of glycosylated haemoglobins using immobilized phenylboronic acid. Effect of ligand concentration, column operating conditions, and comparison with ion-exchange and isoelectric-focusing

Abstract

Haemoglobins from diabetic and non-diabetic individuals have been separated by affinity chromatography using immobilized phenylboronate, which interacts specifically with diol-containing compounds such as glycosylated haemoglobin. The effects of ligand concentration, flow rate, column geometry, preincubation of sample, buffer composition and temperature have been investigated. Significant correlation was found between results from affinity-chromatography and ion-exchange and isoelectric-focusing methods. Isoelectric-focusing of the haemoglobin fractions obtained from affinity chromatography indicate that, in addition to haemoglobin A1c, some haemoglobin A is also bound to immobilized phenylboronic acid. Assays of haemolysates obtained from red blood cells incubated in glucose solutions suggest that unstable pre-(haemoglobin A1c) does not interfere. The assay is not affected by the presence of haemoglobin F.

Glycation of Host Proteins Increases Pathogenic Potential of Porphyromonas gingivalis

“…Following incubation, the hemoglobin was desorbed from the filter paper in the above buffer and residual glucose and buffer constituents were removed by dialysis at 5 °C, firstly against deionized water, then against a 0.25 M ammonium acetate buffer, pH 8.5, and finally against the above buffer containing 20 mM magnesium chloride. The glycated fraction was enriched using phenylboronic acid (PBA) resin (Sigma-Aldrich) affinity chromatography according to Middle et al [ 94 ]. PBA-bound glycated hemoglobin was desorbed from the resin by the addition of 1 mL of 0.25 M ammonium acetate, pH 8.5, containing 0.2 M sorbitol, and then dialyzed against deionized water at 5 °C to remove residual sorbitol.…”

Click Reactions and Boronic Acids: Applications, Issues, and Potential Solutions

“…In organic chemistry, boronic acids are very important in Suzuki-Miyaura coupling [ 1 ], aromatic functionalization (such as amination) with a heteroatom-containing functional group [ 2 ], protection of diols [ 3 ], Diels-Alder reactions [ 4 , 5 ], asymmetric synthesis of amino acids [ 6 ], selective reduction of aldehydes [ 7 ], carboxylic acid activation [ 8 , 9 , 10 ], transition metal-catalyzed asymmetric conjugate additions of boronic acids [ 11 , 12 ], addition to carbonyl and imine derivatives [ 13 , 14 , 15 ], and as a template in organic synthesis [ 16 ]. In materials chemistry, boronic acids are important in crystal engineering [ 17 , 18 ], construction of polymers with reversible properties [ 19 , 20 ], building unique molecular architects [ 21 , 22 , 23 , 24 ], functionalization of nanostructures [ 25 ], separation and purification of glycosylated products [ 26 , 27 ] and feed-back controlled drug delivery (glucose) [ 28 ]. In bioorganic chemistry, boronic acid is a commonly used recognition moiety for the design and synthesis of sensors for carbohydrates [ 29 ], amino acids [ 30 ], amino alcohols [ 31 , 32 ], cyanides [ 33 ], fluoride [ 34 ] and α-hydroxy acids [ 35 , 36 ].…”

Separation of neoglycoproteins with different degrees of glycosylation by boronate chromatography

“…Although examples exist that illustrate the possibility of using this technique to isolate glycoproteins [10 14], most applications focus on the isolation of glycoproteins from non-glycosylated molecules. One of the most widely used applications is in the analysis of the average percentage of glycosylated haemoglobin as a diagnostic tool in detecting and treating diabetes [9,11,14]. In this procedure, glycosylated haemoglobin is isolated from non-glycosylated components, but the glycosylated fraction probably comprises different glycoforms.…”