Metal ion concentration, time, and pH dependence of metal ion binding to a transferrin metalloprotein affinity chromatography (MAMC) matrix

Abstract

The adsorption of transition metal, lanthanide, and actinide ions to ovotransferrin (conalbumin) immobilized to sepharose (via the cyanogen bromide method) has been examined. Adsorption of ions as a function of time and adsorption isotherms at pH 8 have been determined and analyzed using the Freundlich model. Distribution coefficients between the pH values 2 and 9 have been measured. The results indicate that immobilization of the protein has little effect on its interactions with metal ions compared with the protein in solution, an important prerequisite for use of this matrix in metalloprotein affinity metal chromatography (MAMC).Key words: transferrin, conalbumin, adsorption isotherms. NOTATION C,,Metal ion concentration in solution at equilibrium (mg dm-3) EDTA Ethylenediaminetetraacetate Hepes 4-(2-hydroxyethyl)-1-piperazine-2-ethanesulfonic-Metal ion quantity adsorbed on matrix qeq Metal ion quantity adsorbed on maxtrix at equilibrium (mg g-')(mg g-l )methodology depends on the cost and robustness of the support and the p r~t e i n ,~ as well as the cost of the reagents and methods utilized to achieve a functional metal affinity column. In order to optimize the cost of operation, a detailed knowledge of the conditions associated with metal binding and release is required. If metal ion-binding to the supported protein reaches equilibrium rapidly, as is observed for the free protein," then this equilibrium can be represented by the Freundlich adsorption i~o t h e r m ,~ which in its linearized form given below allows for detailed comparison of the capacity ( K , ) and intensity (n) of metal ion binding under a variety of conditions.

Copper binding and release by immobilized transferrin: A new approach to heavy metal removal and recovery

“…where q eq is the uptake of metal at equilibrium, C is the equilibrium solution concentration of metal, K f is the Freundlich constant and ln(K f ) gives a measure of the adsorbent capacity, and n is a constant, where 1/n gives the intensity of adsorption (Adamson, 1976). Cannell and Vincent (1995) have reported n to be 1.28 g/L for Cu 2+ , compared to 0.914 for Fe 3+ . The results indicate that iron binds more tightly to the transferrin/sepharose matrix than does copper.…”

Quantifying metal ion specificity of the nickel-binding proteinCcNikZ-II fromClostridium carboxidivoransin the presence of competing metal ions

“…However, the quantitative relationship between exchange constant and in vitro protein metalation fractions in a multimetal solution remains unclear in general. While, methodologies have been developed to calculate fractions of a protein bound to these different metals in vivo [4,5], a growing area of biotechnology research considers the use of metalloproteins in vitro to purify specific metals from wastewater containing several metals in large excess [22][23][24]. A quantitative grasp of this relationship could thus enable model-guided design of metalloproteins towards more selective biotechnologies in bio-based metal purification.…”