Multiple peaks induced by domain-specific binding of fibrinogen in anion-exchange high-performance liquid chromatography.
A domain binding model was developed for explaining the multiple peak chromatograms obtained in the high-performance liquid chromatography of pure fibrinogen on a DEAE polymethacrylate column using different gradients of ammonium chloride. The different peaks for fibrinogen result from the binding of either the D or E domain of fibrinogen to the packing material. This was confirmed by comparing the retention times of the chromatograms for fibrinogen, fragment D1 and fragment E. Native and denatured forms of fibrinogen are proposed to be important to fibrinogen's interaction with the column, hiding or exposing the E domain, respectively. Different gradient speeds resolve a different number of peaks for fibrinogen, with slow gradients yielding essentially one peak and fast gradients 10 or more peaks. Temperature studies were done to confirm the model. Different commercial sources of fibrinogen showed different proportions of native and denatured/degraded forms.
Liu
Y
Y
Department of Chemistry, Cleveland State University, OH 44115, USA.
Anderson
D J
DJ
Shainoff
J R
JR
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D013816:Thermodynamics
D005340:Fibrinogen