title: Hemoglobin binding from human blood hemolysate with poly(glycidyl methacrylate) beads.
abstract: Metal-chelating affinity beads have attracted increasing interest in recent years for protein purification. In this study, iminodiacetic acid (IDA) was covalently attached to the poly(glycidyl methacrylate) [PGMA] beads (1.6 μm in diameter). Cu(2+) ions were chelated via IDA groups on PGMA beads for affinity binding of hemoglobin (Hb) from human blood hemolysate. The PGMA beads were characterized by scanning electron microscopy (SEM). The PGMA-Cu(2+) beads (628 μmol/g) were used in the Hb binding-elution studies. The effects of Hb concentration, pH and temperature on the binding efficiency of PGMA-Cu(2+) beads were performed in a batch system. Non-specific binding of Hb to PGMA beads in the absence of Cu(2+) ions was very low (0.39 mg/g). The maximum Hb binding was 130.3 mg/g. The equilibrium Hb binding increased with increasing temperature. The negative change in Gibbs free energy (ΔG°<0) indicated that the binding of Hb on the PGMA-Cu(2+) beads was a thermodynamically favorable process. The ΔS and ΔH values were 102.2 J/mol K and -2.02 kJ/mol, respectively. Significant amount of the bound Hb (up to 95.8%) was eluted in the elution medium containing 1.0 M NaCl in 1 h. The binding followed Langmuir isotherm model with monolayer binding capacity of 80.3-135.7 mg/g. Consecutive binding-elution experiments showed that the PGMA-Cu(2+) beads can be reused almost without any loss in the Hb binding capacity. To test the efficiency of Hb depletion from blood hemolysate, eluted portion was analyzed by fast protein liquid chromatography. The depletion efficiency for Hb was above 97.5%. This study determined that the PGMA-Cu(2+) beads had a superior binding capacity for Hb compared to the other carriers within this study.
authors: Hacettepe University, Department of Chemistry, Biochemistry Division, Ankara, Turkey.
authors: Evrim Banu
authors: EB
authors: Altıntas
authors: Deniz
authors: D
authors: Türkmen
authors: Veyis
authors: V
authors: Karakoç
authors: Adil
authors: A
authors: Denizli
mesh_terms: D000465:Algorithms; D001667:Binding, Competitive; D002623:Chemistry Techniques, Analytical / Q000379:methods; D002853:Chromatography, Liquid; D003300:Copper / Q000737:chemistry*; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007098:Imino Acids / Q000737:chemistry*; D007700:Kinetics; D008855:Microscopy, Electron, Scanning; D008863:Microspheres; D008956:Models, Chemical; D015394:Molecular Structure; D011109:Polymethacrylic Acids / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D015203:Reproducibility of Results; D013696:Temperature; D013816:Thermodynamics
chemical_list: D006454:Hemoglobins; D007098:Imino Acids; D011109:Polymethacrylic Acids; C042535:polyglycidyl methacrylate; D003300:Copper; C008109:iminodiacetic acid