Immobilized metal ion affinity chromatography of synthetic peptides. Binding via the alpha-amino group.
Peptides synthesized by the solid-phase method can be efficiently purified in a single immobilized metal affinity chromatography step based on interaction with the alpha-amino group if, after coupling of each amino acid residue, unreacted amino groups are irreversibly blocked by acetylation and if no strongly metal-binding amino acids (His, Trp, Cys) are present in the sequence. A difference in basicity for alpha- and epsilon-amino functions of ca. 2 pH units is sufficiently large to allow selective binding of peptides to immobilized metal ions via the unprotonated alpha-amino group. The binding is pH-dependent: on Cu(2+)- and Ni(2+)-loaded supports most peptides are maximally retarded at pH values around 7.5 and 8.5, respectively. The decreased binding strength at lower pH values is due to protonation of the alpha-amino function, whereas the reduced affinity at higher pH is caused by metal ion transfer from the matrix to the peptide. The metal ion is captured in a multidentate chelate where, in addition to the alpha-amino group, up to three adjacent deprotonated amide nitrogens are coordinated to the metal. If the pH is raised further, additional metal ions may be bound in biuret-like structures. Immobilized Ni2+, owing to its higher selectivity and affinity, is the preferred chromatographic support if slightly basic conditions can be tolerated.
Hansen
P
P
Department of Immunology, University of Uppsala, Sweden.
Lindeberg
G
G
Andersson
L
L
D000595:Amino Acid Sequence; D002021:Buffers; D002413:Cations, Divalent; D002846:Chromatography, Affinity / Q000379:methods*; D003300:Copper / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009532:Nickel / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D014364:Tryptophan / Q000378:metabolism
D002021:Buffers; D002413:Cations, Divalent; D010455:Peptides; D006639:Histidine; D003300:Copper; D009532:Nickel; D014364:Tryptophan; D003545:Cysteine