Chromatographic separations of serum proteins on immobilized metal ion stationary phases.
The separation of proteins on stationary phases consisting of a bound organic chelator and a chelated divalent transition metal has been studied as a function of (A) metal ion species; (B) mobile phase composition and pH; and (C) anion and cation concentration. Optimum separation was observed at alkaline pH on chelated nickel stationary phases. Ammonium and Tris salts reduced the affinity of the metal chelate packing for serum proteins. Halide ions caused the proteins to be more strongly bound to the stationary phase. High salt concentrations had only a small effect on the binding of serum proteins in the absence of amine containing buffers or salts. It was also observed that the ease of elution and the recovery of protein were dependent on pH and upon the presence of halides. The general order of elution of serum proteins, based on isoelectric focusing, was independent of metal ion species and elution conditions, suggesting that a single mechanism or a unique sequence of mechanisms was operative. The results suggest that ligand exchange is the major mechanism of separation under basic conditions and that hydrophobic effects are the result of the competition of nonnitrogen ions with ammonium ions or amines for ligand binding sites modifying or participating in protein binding. Protein binding studies under weak acidic conditions are also presented although the mechanism responsible for protein binding is unclear.
Margolis
S A
SA
Organic Analytical Research Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899.
Fatiadi
A J
AJ
Alexander
L
L
Edwards
J J
JJ
D001797:Blood Protein Electrophoresis / Q000379:methods*; D001798:Blood Proteins / Q000302:isolation & purification*; D002021:Buffers; D002846:Chromatography, Affinity / Q000379:methods; D003035:Cobalt; D003300:Copper; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007098:Imino Acids; D007525:Isoelectric Focusing; D008345:Manganese; D008670:Metals; D009532:Nickel; D009994:Osmolar Concentration; D012492:Salts; D012685:Sepharose; D015032:Zinc
D001798:Blood Proteins; D002021:Buffers; D007098:Imino Acids; D008670:Metals; D012492:Salts; D003035:Cobalt; D008345:Manganese; D003300:Copper; D009532:Nickel; D012685:Sepharose; D015032:Zinc; C008109:iminodiacetic acid