Active sites antigens

The powerful inhibition of Ca " transport without inhibition of ATPase activity seen with polyclonal anti-ATPase sera in earlier studies [305] was probably due to complement dependent lesion of the membrane that permitted the leakage of accumulated calcium [306-308], The scarcity of inhibitory antibodies may imply that the active site of the Ca - ATPase is either inaccessible to antibodies or poorly antigenic, perhaps due to a unique secondary structure. 

One of the best ways to ensure retention of activity in protein molecules is to avoid doing chemistry at the active center. The active center is that portion of the protein where ligand, antigen, or substrate binding occurs. In simpler terms, the active center (or active site) is that part that has specific interaction with another substance (Means and Feeney, 1971). For the preparation of enzyme derivatives, it is important to protect the site of catalysis where conversion of substrate to product happens. For instance, when working with antibody molecules, it is crucial to stay away from the two antigen binding sites. 

The most common of these systems is the enzyme-multiplied immunoassay technique or EMIT, which is particularly suited to the measurement of small molecules (haptens) such as drugs. EMIT is a trade mark of the Syva Corporation of Palo Alto, California. Although it does not involve the separation of bound fraction from free it is nevertheless a competitive assay system. The antigen is labelled with an enzyme in such a way that the enzyme retains its catalytic activity. When the antigen binds to the antibody the enzyme becomes inhibited, probably by an induced conformational change or by steric hindrance of the enzyme active site (Figure 7.15). 

Molecular recognition in biological systems (active sites on the surfaces of macromolecule, antibody-antigen) and biological sensors (enzyme activity, biosensors). 

Subunits do not form a precipitate with antiphosphatase antibody however, there appear to be some antigenic determinates common to both subunits and active enzyme since subunits interfere with the precipitation of alkaline phosphatase. The alkaline phosphatase-antibody complex has 70% of the original enzymic activity as a suspension in solution. Therefore, the antibody does not bind to the active site of alkaline phosphatase, but it can still differentiate between monomers and dimers. 

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