The interactive behavior of an amphipathic peptide with the Cu2+, Ni2+, and Zn2+ complexes of 1,4‐bis(triazacyclonon‐1‐yl)butane), bis(tacn)but, immobilized onto Sepharose CL‐4B, has been investigated. The effects of incubation time, as well as the incubation buffer pH and ionic strength, have been examined. The binding data have been interrogated using Langmuir, Langmuir‐Freundlich, bi‐Langmuir, and Temkin isothermal models and Scatchard plots. These results confirm that this amphipathic peptide binds with relatively high capacities to the immobilized Cu2+‐ and Ni2+‐1,4‐bis(triazacyclonon‐1‐yl)butane)‐Sepharose CL‐4B sorbents via at least two discrete sites. However, the corresponding immobilized Zn2+‐sorbent had low binding capacity. Moreover, the magnitude of the binding capacities of these sorbents was dependent on the pH and ionic strength of the incubation buffer. These results are relevant to the isolation of E. coli expressed recombinant proteins that incorporate this and related amphipathic peptide tags, containing two or more histidine residues, located at the N‐ or C‐terminus of the recombinant protein, and the co‐purification of low abundance host cell proteins of diverse structure, by immobilized metal ion affinity chromatographic methods.
The X-ray crystal structures of 1,4,7-tris(methylenephosphonate)-1,4,7,10-tetraazacyclododecane (DO3P·3.5H2O) and 1,7-bis(methylenephosphonate)-1,4,7,10-tetraazacyclododecane dihydrochloride trihydrate (DO2P·2HCl·3H2O) reveal that two nitrogen atoms in the trans-annular positions are protonated. The macrocyclic ring adopts a (3,3,3,3)-B conformation stabilized by intramolecular hydrogen bonding involving oxygen atoms from the phosphonate groups and the protonated amines. The acid–base dissociation constants of DO3P and metal complex formation constants (for Ca2+, Mg2+, Zn2+, Fe3+) were determined using potentiometric measurements. The first two protonations of DO3P occur at quite high pH and, consequently, were determined by NMR measurements. The stability constants indicate that DO3P forms more stable complexes with the transition metal ions Zn2+ and Fe3+ (log K
[ML] = 21.23(4) and 22.74(5) respectively) than with the alkaline earth metal ions Ca2+ and Mg2+ (log K
[ML] = 10.96(6) and 8.72(6) respectively). For Fe3+, Ca2+ and Mg2+, the data support the formation of dinuclear species, presumably through the coordination of an additional metal ion to the phosphonate groups.
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