Study of haptoglobin-hemoglobin complexes by titration curves, capillary electrophoresis and capillary isoelectric focusing.
A novel method is described for monitoring complex formation between macromolecules, based on combined isoelectric focusing-electrophoresis in capillaries. The example studied is the binding of serum haptoglobin (Hp) to hemoglobin (Hb). A known amount of Hb is focused in a capillary in a pH 6-8 range (pI of Hb = 7.0) and thus kept temporarily "immobilized" in the electrophoretic chamber. Subsequently, increasing amounts of ligand (Hp) are loaded cathodically and allowed to sweep past the focused Hb zone. As the complex formed has a pI value well-outside the bounds of such a pH gradient (the 1:1 molar Hb-Hp complex has a pI of 5.5, the 1 to 1/2 molar Hp-Hb complex has a pI of 5.0) it escapes immobilization and moves past the detector window, where it is monitored and quantified. Since the detector is set at 416 nm, where only Hb absorbs, and since the molar extinction coefficient of Hb is well known, it is quite easy to calculate the molar amount of Hb bound to the complex. As an additional check, the amount of unreacted Hb can now be mobilized by disrupting the pH gradient and allowing this residual free Hb to also reach the detector and be quantified. The method is easy, fast, simple and fully automated and thus could represent a valid alternative to existing methods in clinical chemistry for quantifying the amount of Hp in human sera in pathological conditions, such as hemolytic anemias and transfusion reactions.
Righetti
P G
PG
Department of Agricultural and Industrial Biotechnology, University of Verona, Italy.
Conti
M
M
Gelfi
C
C
D000665:Ampholyte Mixtures; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D006242:Haptoglobins / Q000032:analysis*; D006454:Hemoglobins / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D017186:Titrimetry
D000665:Ampholyte Mixtures; D006242:Haptoglobins; D006454:Hemoglobins; C035492:haptoglobin-hemoglobin complex