Affinity macroligand

Stocker-Majd, G., Hilbrig, F. and Freitag, R. (2008). Extraction of haemoglobin from human blood by affinity precipitation using a haptoglobin-based stimuli-responsive affinity macroligand.of Chromatography A, 1194, 57-65. 

Figure 3.2. Schematic presentation of a secondary effect affinity precipitation using a heterobifunctional affinity macroligand to precipitate the target molecule (reproduced with permission from reference 25).

Freitag R. Reversibly water-soluble affinity macroligands for bioseparation. Curr Trends Polym Sci 1998 3 63-79. 

This possibility of calculating the minimum, though sufficient, amount of macroligand to complex the desired biomolecule, affects the quality of the subsequent purification. In fact, the use of minimum quantities of affinity macroligand - for instance, 25 mg of dextran-E for the purification of 10 ml of the 4S-trypsin calf uterus estradiol receptor preparation [20] - enables one to reduce to a very low level the nonspecific adsorption of contaminants on the affinity material. Compared to affinity chromatography, where gram amounts of the matrix are frequently required, this low contamination at the association stage must obviously lead to better purification ratios. 

Figure 11 Schematic representation of techniques used for solubilized and immobilized affinity ligands in polyacrylamide gels. (A) Solubilized ligand method the ligand can move freely in the gel-buffer system. (B) Macroligand method the solution of acrylamide contains the macroligand that becomes entrapped within the gel matrix after polymerization. (C) Chemically bound ligand direct copolymerization of polyacrylamide gel with the copolymerizable derivative of the ligand. (Reproduced with permission from Ref. 13.)...

The most common way of preparing affinity-electrophoresis immobilized ligands is to synthesize a water-soluble macromolecular derivative of the ligand, called a macroligand. This is currently achieved by copolymerization of an acryloyl derivative of the ligand with acrylamide or a related monomer. 

Preparation of affinity gels T = 6 per cent) with entrapped macroligand... 

For 1200 xl of affinity gels, progressively increase the volume of macroii-gand solution up to 300 jxl at the expense of water. Some macroligands may inhibit acrylamide polymerization if this occurs, increase the concentration of polymerization catalysts. 

The recovery was 50-75%, with respect to the amount of receptor present in the aggregate-free preparation (depending on the initial quality of the calf uterus tissue), and the purity was estimated to about 80%. These results were quite competitive with those afforded by traditional affinity chromatography [22,23], if not better, and were obtained in a short time without previous experience in the field of estrogen receptor purification. The quantity of material that could be treated by this technique was only limited by the size of the gel filtration columns. On the other hand, the water-soluble macroligand could be recovered in the void volume of the column, at the second filtration step, and recycled for further purification experiments, after removal of the non-specifically bound contaminants. 

In one of the earliest studies [39], we carried out the purification of A5 4 3-oxosteroid isomerase from a crude extract of Pseudomonas testosteroni by affinity pardoning in a poly(ethylene glycol)/dextran system. The macroligand was prepared by covalent coupling on poly(ethylene glycol) (PEG) of the same steroid derivative as previously described in Fig. 14.2. The resulting macroligand PEG-E,i had an inhibition constant Ki around 5 pM towards the isomerization of A5-androstene-3,17-dione, by isomerase. 

Poly(ethylene glycol) is the most widely used polymer to prepare macroligands for affinity partition. This is probably partly due to its solubility both in... 

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