Synthetic Ligands

Textile dyes had already proved to be suitable ligands for protein separations. Blood proteins, dehydrogen-ases, kinases, oxidases, proteases, nucleases, transfer-ases and ligases can be purified by a wide variety of dyes. However, they did not prove to be the breakthrough so eagerly awaited. An essential feature of all chromatographic processes is exact repeatability from column to column, year after year. Textile dyes are bulk chemicals, most of which contain many by-products, co-produced at every stage of the dye

Figure 3 Comparison of multistep versus affinity separation.

manufacturing process. This fact alone makes reproducibility problematic. Furthermore, the bonding process between dye and matrix was poorly researched. This resulted in extensive leakage. All commercially available textile dye products leak extensively, especially under depyrogenating conditions (Figure 4). Despite these limitations, it was recognized that dye-like structures had a powerful underlying ability to separate a very diverse range of proteins. Their relatively complex chemical structures allow spatial manipulation of their basic skeletons into an infinite variety of shapes and configurations. Proteins are complex three-dimensional (3-D) structures and folds are present throughout all protein structures. An effective ligand needs to be shaped in such a manner that it allows deep insertion into a suitable surface fissure existing within the 3-D structure (Figure 5). In contrast, if the ligand only interacts with groups existing on external surfaces, then nonspecific binding results and proteins other than the target are also adsorbed. A much more selective approach is to attempt to insert a ligand into an appropriate fold of the protein, and add binding groups to correspond with those present in a fold of the protein. If all four of the basic intermolecular forces (Figure 5: W, electrostatic; X, hydrogen bond-

Ligand Protein Interaction

Figure 5 Schematic representtion of ligand-protein interaction. W, electrostatic interaction; X, hydrogen bonding; Y, van der

Waals interaction; Z, hydrophobic interaction. -, original backbone; —, new structure added; •••, original backbone move; < >, fields of interaction.

Figure 5 Schematic representtion of ligand-protein interaction. W, electrostatic interaction; X, hydrogen bonding; Y, van der

Waals interaction; Z, hydrophobic interaction. -, original backbone; —, new structure added; •••, original backbone move; < >, fields of interaction.

ing; Y, van der Waals; Z, hydrophobic) align with the binding areas in the protein fold, idealized affinity reagents result. The use of spacer arms minimizes steric hindrance between the carrying matrix and protein.

Figure 4 Leakage of blue dye from various commercial products. □, 0.1 mol L~1 NaOH; H, 0.25 mol L~1 NaOH; ■, 1 mol L~1 NaOH. Key: A, Mimetic Blue 1 A6XL (affinity chromatography); B, Affi-Gel Blue (Bio-Rad); C, Blue Trisacryl-M (IBF); D, Fractogel TSK AF-Blue (Merck); E, C.I. Reactive Blue 2 polyvinyl alcohol-coated perfluropolymersupport; F, Blue Sepharose CL-6B (Pharmacia); G, immobilized Cibacron Blue F3G-A (Pierce); H, Cibacron Blue F3G-A = Si500 (Serva); I, Reactive Blue 2-Sepharose CL-6B (Sigma).

Figure 4 Leakage of blue dye from various commercial products. □, 0.1 mol L~1 NaOH; H, 0.25 mol L~1 NaOH; ■, 1 mol L~1 NaOH. Key: A, Mimetic Blue 1 A6XL (affinity chromatography); B, Affi-Gel Blue (Bio-Rad); C, Blue Trisacryl-M (IBF); D, Fractogel TSK AF-Blue (Merck); E, C.I. Reactive Blue 2 polyvinyl alcohol-coated perfluropolymersupport; F, Blue Sepharose CL-6B (Pharmacia); G, immobilized Cibacron Blue F3G-A (Pierce); H, Cibacron Blue F3G-A = Si500 (Serva); I, Reactive Blue 2-Sepharose CL-6B (Sigma).

The final step is to design appropriate bonding technologies to minimize potential leakage. Until recently this type of modelling was a purely theoretical exercise. It was only the introduction of computerassisted molecular modelling techniques that allowed the theory to be tested. Before the arrival of logical modellling the discovery of selective ligands was entirely based upon empirical observation, later followed by a combination of observation, experience and limited assistance from early computer generated models. Although several novel structures evolved during this period, a general approach to the design of new structures remained elusive. At this time only very few 3-D protein structures were available, again greatly restricting application of rational design approaches. As more sophisticated programmes, simulation techniques, protein fragment data and many more protein structures were released, logical design methods were revolutionized. However, many millions of proteins are involved in life processes, and it is clear that many years will elapse before the majority of these will be fully described by accurate models. Consequently intuition and experience will continue to play a major role in the design of suitable ligands. Of available rationally designed synthetic molecules, the Mimetic™ range can currently separate over 50% of a randomly selected range of proteins. Stability under depyrogenating conditions has been demonstrated for these products (Figure 6). This results in minimal contamination from ligand and matrix im-

Figure 6 Comparison of ligand leakage from mimetic ligand affinity adsorbent A6XL (#) and conventional textile dye agarose

Figure 6 Comparison of ligand leakage from mimetic ligand affinity adsorbent A6XL (#) and conventional textile dye agarose

purities, substantial increases in column lifetime, and improvements in batch-to-batch reproducibility.

Solar Panel Basics

Solar Panel Basics

Global warming is a huge problem which will significantly affect every country in the world. Many people all over the world are trying to do whatever they can to help combat the effects of global warming. One of the ways that people can fight global warming is to reduce their dependence on non-renewable energy sources like oil and petroleum based products.

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