Evaluation and optimization of the metal‐binding properties of a complex ligand for immobilized metal affinity chromatography

Abstract

The simultaneous determination of two binding parameters for metal ions on an immobilized metal affinity chromatography column was performed by frontal chromatography. In this study, the binding parameters of Cu(2+) to l-glutamic acid were measured, the metal ion-binding characteristics of the complex ligand were evaluated. The linear correlation coefficients were all greater than 99%, and the relative standard deviations of two binding parameters were 0.58 and 0.059%, respectively. The experiments proved that the frontal chromatography method was accurate, reproducible, and could be used to determine the metal-binding parameters of the affinity column. The effects of buffer pH, type, and concentration on binding parameters were explored by uniform design experiment. Regression, matching and residual analyses of the models were performed. Meanwhile, the optimum-binding conditions of Cu(2+) on the l-glutamic acid-silica column were obtained. Under these binding conditions, observations and regression values of two parameters were similar, and the observation values were the best. The results demonstrated that high intensity metal affinity column could be effectively prepared by measuring and evaluating binding parameters using frontal chromatography combined with a uniform design experiment. The present work provided a new mode for evaluating and preparing immobilized metal affinity column with good metal-binding behaviors.

Uniform experimental design in chemometrics

“…Ji, Alaert, and Xu et al proposed sequential UDs to seek the best extracts through the capillary electrophoresis fingerprint of G. biloba . There were other works that used UD to optimize the experimental conditions for good separation to analyze chemical components or to obtain high experimental yields …”

Rapid and efficient one‐step purification of a serralysin family protease by using a p‐aminobenzamidine‐modified affinity medium

“…According to the Scatchard method, the data should fit the following equation (Eq. ) : …”

“…To characterize the affinity value of the enzyme and the synthesized affinity material, equilibrium adsorption characteristics were evaluated. The K d (the constant of desorption) and Q max (the theoretical maximum adsorption capacity) of the affinity medium were analyzed according to the Scatchard analysis model . Briefly, 1 mL of different concentration solutions of purified MP (ranging 0.1–0.9 mg/mL in 100 mM Tris titrated by HCl to pH 7.6) were blended with 0.5 g of pABA‐modified material and shaken for 2 h at 25°C until the solution reached the adsorption equilibrium.…”

Structure-Based Design and Synthesis of a New Phenylboronic-Modified Affinity Medium for Metalloprotease Purification

“…Although some reports refer to the high-yield purification of metalloproteases (more than 90% purity) in one-step procedure, these protocols were based on immobilized metal affinity chromatography (IMAC) that has its disadvantages [ 21 , 22 ]. The first one is the use of high concentrations of imidazole and salt in the elution buffer of the IMAC procedure, which necessitates additional dialysis or a desalting step [ 23 , 24 ]. Also, it is well known that purification of a metalloprotein via a metal ion chelated by the resin in a similar manner results in the exchange of metal ion from resin with a metal ion from metalloprotein.…”

“…This metal transfer causes a decrease in the stability of purified metalloprotein [ 21 ]. In addition, the use of chelating agents during purification has to be avoided as these compounds can remove the metal ion from the enzyme active site [ 23 , 24 , 25 ]. Therefore, design, synthesis and application of a new specific and efficient medium for the purification of metalloproteases are important tasks.…”