Native immobilizing reagents

Until recently, the catalytic role of Asp ° in trypsin and the other serine proteases had been surmised on the basis of its proximity to His in structures obtained from X-ray diffraction studies, but it had never been demonstrated with certainty in physical or chemical studies. As can be seen in Figure 16.17, Asp ° is buried at the active site and is normally inaccessible to chemical modifying reagents. In 1987, however, Charles Craik, William Rutter, and their colleagues used site-directed mutagenesis (see Chapter 13) to prepare a mutant trypsin with an asparagine in place of Asp °. This mutant trypsin possessed a hydrolytic activity with ester substrates only 1/10,000 that of native trypsin, demonstrating that Asp ° is indeed essential for catalysis and that its ability to immobilize and orient His is crucial to the function of the catalytic triad. 

It is also worthwhile to outline at this place the immobilization procedure that was used for the preparation of type I CSPs A bifunctional linker with a terminal isocyanate on one side and a triethoxysilyl group on the other end (3-isocyanatopropyl triethoxysilane) was reacted with the native cinchona alkaloids quinine and quinidine and subsequently the resultant carbamate derivative in a second step with silica [30], Remaining silanols have been capped with silane reagents, yet, are less detrimental for acidic solutes because of the repulsive nature of such electrostatic interactions. CSPs prepared in such a way lack the hydrophobic basic layer of the thiol-silica-based CSPs mentioned earlier, which may be advantageous for the separation of certain analytes. 

It presents a comparison of the observed GOD activities with activity of GOD physisorbed on native, underivatized CPG. An increase of enzyme activity over a time of four weeks was observed. The first bar shows the absorption of the sample immobilized without any reagent on native CPG. The second bar results from immobilization using APTES/GA, and the last two bars show the activity of GOD immobilized using aldehyde-functionalized silanes with the same chain length (C7) but differing in the number of substituent efrioxy groups. 

The reverse capture autoantibody microarray is based on the dual-antibody sandwich immunoassay of enzyme-linked immunosorbent assay (ELISA) (see Fig. 1). The basic platform consists of a glass microarray slide arrayed with 1000 highly specific well-characterized monoclonal antibodies against 500 unique antigens. These antibodies are used to immobilize native proteins. Because the reagents used in the procedure are non-denaturing, antigens are... 

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