Interaction of plasma proteins with heparinized gel particles studied by high-resolution two-dimensional gel electrophoresis.
In order to further the understanding of protein-surface interactions in the coagulation system, we have chosen to study plasma protein adsorption onto heparin-immobilized surfaces. Heparin-binding proteins are abundant in plasma: a search of amino acid sequences revealed that many plasma proteins have possible heparin binding sites. Plasma protein adsorption to the heparinized surfaces is monitored by a novel technique in which the solution depletion of proteins is analytically determined using quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). This method enables simultaneous, quantitative detection of the majority of plasma proteins before, during, and after their adsorption onto high surface area adsorbents. Using computerized densitometry of silver-stained 2-D PAGE gels, the amount of each protein can be determined from the integrated optical density of each protein "spot." Kinetics of adsorption and adsorption isotherms of four important heparin binding proteins, antithrombin III (ATIII), complement factor C3 (C3), apolipoprotein AI (Apo-AI) and apolipoprotein AIV (Apo-AIV) are reported in this paper. From the adsorption isotherms, the apparent binding constants of each protein-immobilized heparin complex, Ka, were calculated. The surface binding constants were of the same order of magnitude as the respective solution binding constants in the literature. The surface binding constants followed the same order as the respective solution binding constants: Ka (ATIII) greater than Ka (Apo-AIV) greater than Ka (C3) greater than Ka (Apo-AI), indicating that protein binding to the immobilized heparin used is not essentially different from solution binding.
Ho
C H
CH
Department of Materials Science & Engineering, University of Utah, Salt Lake City 84112.
Hlady
V
V
Nyquist
G
G
Andrade
J D
JD
Caldwell
K D
KD
D000327:Adsorption; D000595:Amino Acid Sequence; D001053:Apolipoproteins / Q000737:chemistry; D001798:Blood Proteins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005782:Gels / Q000737:chemistry*; D006493:Heparin / Q000737:chemistry*; D006801:Humans; D007525:Isoelectric Focusing; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D013194:Staining and Labeling; D013816:Thermodynamics
D001053:Apolipoproteins; D001798:Blood Proteins; D005782:Gels; D006493:Heparin