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.
[Show abstract][Hide abstract] ABSTRACT: Generated by proteolytic cleavage of immunoglobulin, Fab fragments possess great promise as blocking reagents, able to bind receptors or other targets without inducing crosslinking. However, aggregation of Fab preparations is a common occurrence, which generates intrinsic stimulatory capacity and thwarts signal blockade strategies. Using a panel of biochemical approaches including size-exclusion chromatography (SEC), SDS-PAGE, mass spectrometry, and cell stimulation followed by flow cytometry, we have measured the oligomerization and acquisition of stimulatory capacity that occurs in four monoclonal IgG Fabs specific for TCR/CD3. Unexpectedly, we observed that all Fabs spontaneously formed complexes that were precisely bivalent, and these bivalent complexes possessed most of the stimulatory activity of each Fab preparation. Fabs composing bivalent complexes were more susceptible to proteolysis than monovalent Fabs, indicating a difference in conformation between the Fabs involved in these two different states of valency. Because osmolytes represent a class of compounds that stabilize protein folding and conformation, we sought to determine the extent to which the amino acid osmolyte L-proline might impact bivalent Fab complexation. We found that L-proline (i) inhibited the adoption of the conformation associated with bivalent complexation, (ii) preserved Fab monovalency, (iii) reversed the conformation of pre-formed bivalent Fabs to that of monovalent Fabs, and (iv) separated a significant percentage of pre-formed bivalent complexes into monovalent species. Thus, Fab fragments can adopt a conformation that is compatible with folding or packing of a bivalent complex in a process that can be inhibited by osmolytes.
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