Bovine serum albumin catalytic activity

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

Native bovine serum albumin has the surprising property of catalyzing the decomposition of the Meisenheimer complex 1,1-dihydro-2,4,6-trinitrocylohexadienate. Taylor and Silver (1976) prepared albumin fragments 1-306 and 307-581 and separated them without disulfide reduction. These were called native fragments. Neither of these fragments alone has catalytic activity to decompose the Meisenheimer complex, but when mixed together stoichiometrically, 35%... 

Any detectable effect on the reaction or behavior of a particular system by the interior wall of the container or reaction vessel. Because proteins can form high-affinity complexes with glass and plastic surfaces, one must exercise caution in the choice of reaction kinetic conditions. Wall effects can be discerned if one determines catalytic activity under different conditions that minimize or maximize contact of the solution with the container. In principle, an enzyme-catalyzed reaction should proceed at the same rate if placed in a capillary or a culture tube however, contact with the wall is maximized in a capillary, and wall effects should be more prominent. Some investigators add bovine serum albumin to prevent adsorption of their enzyme onto the container s walls. 

Concerning the catalytic superoxide dismutase activity of low molecular mass copper complexes, some general comments are neccessary. Firstly, it should be emphasized, that apart from the inactive Cu-penicillamine, all complexes described above do not survive high concentrations of biological chelators in aqueous solutions. For example, bovine serum albumine is able to remove most of the copper from these complexes (Fig. 14). 

An interesting conformation-dependent catalytic activity of bovine serum albumin (BSA), sheep albumin, and horse albumin has been reported (Taylor and Vatz, 1973), namely the acceleration of decomposition (by a factor of 10 of the Meisenheimer complex 1,1-dihydro-2,4,6-trin-itrocyclohexadienate. The physiological importance of this activity, however, is presently not known and is not exhibited by human serum albumin. 

The approach using cyclodextrin as a binding site has also been developed. Cyclodextrins are widely utilized in biomimetic chemistry as simple models for an enzyme because they have the ability to form inclusion complexes with a variety of molecules and because they have catalytic activity toward some reactions. Kojima et al. (1980, 1981) reported the acceleration in the reduction of ninhydrin and some dyes by a 1,4-dihydronicotinamide attached to 3 Cyclodextrin. Saturation kinetics similar to enzymatic reactions were observed here, which indicates that the reduction proceeds through a complex. Since the cavity of the cyclodextrin molecule has a chiral environment due to the asymmetry of D-glucose units, these chiralities are expected to be effective for the induction of asymmetry into the substrate. Asymmetric reduction with NAD(P)H models of this type, however, has not been reported. Asymmetric reduction by a 1,4-dihydronicotinamide derivative took place in an aqueous solution of cyclodextrin (Baba et al. 1978), although the optical yield from the reduction was quite low. Trifluoromethyl aryl ketones were reduced by PNAH in 1.1 to 5.8 % e.e. in the presence of 3-cyclodextrin. Sodium borohydride works as well (Table 18). In addition to cyclodextrin, Baba et al. also found that the asymmetric reductions can be accomplished in the presence of bovine serum albumin (BSA) which is a carrier protein in plasma. 

It was necessary to carry out enzyme assays over an extended period of time, using the same dilution of enzyme, therefore conditions were sought to minimize time-dependent changes in catalytic power. Dilution of the enzyme in assay buffer alone(10 mM sodium phosphate, pH 6.2) caused approximately 20% decrease in catalytic activity after incubation at 4 C for 4 h. This loss was ameliorated by a combination of raising the ionic strength of the enzyme dilution buffer to 37.5 mM and including Bovine Serum Albumin(BSA 1 mg/ml) DTT(1 mM) EDTA(1 m and glycerol(20% w/v). When this solution was used to dilute the enzyme preparation, full stability was retained over a 5 h period and 98% of the activity, present immediately after dilution, was observed more than 24 h later. 

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