Coupling of m-aminophenylboronic acid to s-triazine-activated Sephacryl: use in the affinity chromatography of glycated hemoglobins

Abstract

An improved process is described for covalent coupling of m-aminobenzeneboric acid to s-triazine-activated Sephacryl matrices. The derivatized Sephacryl gel contained up to 150–200 μmol boronate per ml. It has been applied to the separation of glycated and non-glycated hemoglobins (Hbs) present in red-cell hemolysate. The new bioaffinity support was evaluated by the analysis of 67 diabetic patients and 20 normal adults. The mean value for glycated Hb was 6.6 ± 0.8% for non-diabetics and 11.2 ± 2.9% for diabetics. The method effects group-specific separation between glycated and non-glycated Hbs even in presence of foetal Hb and abnormal Hb variants. There is an excellent correlation between the glycated Hb levels obtained by the new method and two established procedures, namely high-performance liquid chromatography ( r = 0.933) and affinity Merckotest ( r = 0.991). The inter-assay and intra-assay coefficient of variations of less than 3.0% suggest that the method is reproducible. The results indicate that the method may serve as an alternative procedure for the study of glycated proteins. The s-triazine-activated Sephacryl could also be used for immobilizing enzymes and for preparing biospecific absorbents.