Effects of in vitro glycation on Fe3+ binding and Fe3+ isoforms of transferrin.
BACKGROUND
In diabetes, protein function is altered by glycation, but the impact on the Fe3+ binding and antioxidant functions of transferrin (Tf) is largely unknown. The aim of the present study was to investigate the effects of glycation on the distribution of Fe3+ on the two Fe3+ -binding sites of Tf.


METHODS
In vitro glycation of Tf was accomplished by preincubation with glucose for 14 days. Tf was loaded with Fe3+ compounds to achieve theoretical Tf Fe3+ saturations of 32%, 64%, and 96% (monitored by spectrophotometry). Fe3+ -Tf isoforms were separated by isoelectric focusing.


RESULTS
Fe3+ binding was highest when Tf was incubated with Fe:nitrilotriacetic acid and reached a steady state overnight. Increasing the Fe3+ load led to a shift of isoform profile toward the diferric form (Fe2-Tf): in freshly prepared Tf, Fe2-Tf represented 6%, 30%, and 66% of all isoforms at 32%, 64%, and 96% theoretical Fe3+ saturation, respectively. Fe3+ was equally distributed to the monoferric Tf forms with Fe3+ bound to the amino (Fe1N-Tf) and carboxy termini (Fe1C-Tf). Glycation decreased binding of Fe3+ to Tf (monitored at 450 nm). At low theoretical Fe3+ saturation (32%), glycation increased the mean (SD) proportion of Fe2-Tf: 18 (3)% in the presence of 33.3 mmol/L glucose vs 12 (4)% with 0 mmol/L glucose (P = 0.01). In contrast, at 96% theoretical Fe3+ saturation, Fe2-Tf decreased linearly with increasing glycation (r = 0.97; P = 0.008). Preincubation, independent of glycation, favored the Fe1N-Tf isoform at 64% theoretical Fe3+ saturation [27 (0.7)% vs 23 (1.1)% of the Fe1C-Tf isoform; P = 0.009].


CONCLUSIONS
Glycation impairs Fe3+ binding and affects Fe3+ -Tf isoform distribution depending on concentration. The diagnostic implications of these results need further elucidation in clinical studies.
Van Campenhout
Ann
A
Laboratory of Endocrinology, Antwerp Metabolic Research Unit, University of Antwerp, Wilrijk, Belgium.
Van Campenhout
Christel
C
Lagrou
Albert René
AR
Manuel-Y-Keenoy
Begoña
B
D005290:Ferric Compounds / Q000378:metabolism*; D019270:Fructosamine / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007525:Isoelectric Focusing; D020033:Protein Isoforms; D014168:Transferrin / Q000378:metabolism*
D005290:Ferric Compounds; D020033:Protein Isoforms; D014168:Transferrin; D019270:Fructosamine; D005947:Glucose