Structural and functional study of rabbit polyclonal antibody for immunoassay purposes.
ABSTRACT In this study, we investigated the effects of some denaturants, such as urea and heat, on structure and function of rabbit polyclonal antibody and its Fab fragments. Thermal unfolding studies by circular dichroism of antibody and Fab fragments showed that in acidic pH, antibody has multi-transitions whereas Fab fragments have one transition curve; however in neutral pH, thermal unfolding of both had one transition. Effects of urea on the structure of antibody and Fab were studied through fluorescence spectroscopy. Despite exposure of protein to high concentration of denaturant, partial unfolding occurred in both antibody and Fab, but the denaturation of Fab was more considerable than that of antibody. Functional studies indicated that urea and heat causes a decrease in affinity in both antibody and Fab, but deactivation of Fab is more considerable in comparison with the antibody molecule. Turbidity study of antibody and Fab showed that aggregation of Fab occurred at lower temperatures than that of antibody. Our results indicate that Fab has higher sensitivity in comparison with antibody in the unfolding, deactivation, and aggregation processes. Therefore, our data proposes a stabilizing role for Fc.
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ABSTRACT: Thermal unfolding monitored by spectroscopy or calorimetry is widely used to determine protein stability. Equilibrium thermodynamic analysis of such unfolding is often hampered by its irreversibility, which usually results from aggregation of thermally denatured protein. In addition, heat-induced protein misfolding and aggregation often lead to formation of amyloid-like structures. We propose a convenient method to monitor in real time protein aggregation during thermal folding/ unfolding transition by recording turbidity or 90 degrees light scattering data in circular dichroism (CD) spectroscopic experiments. Since the measurements of turbidity and 90 degrees light scattering can be done simultaneously with far- or near-UV CD data collection, they require no additional time or sample and can be directly correlated with the protein conformational changes monitored by CD. The results can provide useful insights into the origins of irreversible conformational changes and test the linkage between protein unfolding or misfolding and aggregation in various macromolecular systems, including globular proteins and protein-lipid complexes described in this study, as well as a wide range of amyloid-forming proteins and peptides.Protein Science 04/2006; 15(3):635-9. · 2.74 Impact Factor
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ABSTRACT: A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.Analytical Biochemistry 06/1976; 72:248-54. · 2.58 Impact Factor
- Biochemistry 12/1968; 7(11):3958-64. · 3.38 Impact Factor