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  • Title: Glycosylation of human hemoglobin A. Kinetics and mechanisms studied by isoelectric focusing.
    Author: Mortensen HB, Christophersen C.
    Journal: Biochim Biophys Acta; 1982 Sep 22; 707(1):154-63. PubMed ID: 7138875.
    Abstract:
    The reaction between glucose and hemoglobin A (HbA) leading to hemoglobin A1c through a labile intermediate, designated anodal glycohemoglobin A, was studied in vitro using an isoelectric focusing method. Studies were performed on the kinetics of the formation and breakdown of the labile intermediate. The reactions of HbA with various aldohexoses and of hemoglobin A1c and anodal glycohemoglobin A with phenylhydrazine were studied. It is suggested that the glucose in the anodal glycohemoglobin A band is in the pyranose form of a Schiff base formed between the N-terminal amino group of the protein and D-glucose, while the fructose in hemoglobin A1c is in the pyranose form of the ketimine comprising also the N-terminal of the group. Elution peaks from ion-exchange chromatography of the hemoglobins termed HbA1a, HbA1b, HbA1c and HbA studied by isoelectric focusing revealed that: HbA1a formed two bands and HbA1b one band far toward the anode; both contained minor fractions of HbAlc and HbA; HbAlc appeared as a single band, while HbA was contaminated with some HbA1c. Hemoglobulin A1c purified by isoelectric focusing eluted as one peak when analysed by ion-exchange chromatography, while anodal glycohemoglobin A co-eluted with the first HbA1c fractions in the chromatogram. From kinetic studies it appeared that the rate constant for formation (k1) of anodal glycohemoglobin A was 0.096 . 10(-3) l/mol . s-1 at 37 degrees C. The constant for dissociation (k-1) was 0.10 . 10(-3) s-1. From these an equilibrium constant K of 0.96 l/mol was calculated. The apparent Arrhenius activation energies for the (k1) and (k-1) reactions were 42.5 and 47.5 kJ/degree, respectively. Consequently the equilibrium constant K is predicted to be nearly temperature-independent within the temperature range investigated. This was fully substantiated by experiments conducted at different temperatures. Furthermore, the values of the apparent Arrhenius activation energies allows the values of the rate constants to be calculated at any temperature within the experimental temperature range. This information is of importance for a closer understanding of the mechanisms of glycohemoglobin accumulation in red blood cells.
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