A Thermodynamic Study of Cooperative Structures in Rabbit Immunoglobulin G

Abstract

The intramolecular melting of rabbit immunoglobulin G and its proteolytic fragments has been studied by scanning microcalorimetry in the acidic pH region. By thermodynamic analysis of the heat capacity function the following conclusions have been made. The Fab and Fc fragments in IgG molecules are thermodynamically practically independent subunits in the pH region studied. The Fab fragment exhibits three cooperative transitions at melting. Two of these transitions are explained assuming that equivalent domains in this fragment are paired, forming two cooperative blocks, while the third transition seems to correspond to the disruption of contacts between these cooperative blocks. Fc and pFc' fragments exhibit four and two cooperative transitions, respectively. These transitions can be explained only by assuming that each of the structural domains of these fragments consists of two subparts and that the pairs of equivalent subparts are merged into the cooperative blocks thus forming four cooperative blocks in the Fc fragment.

The use of native cation‐exchange chromatography to study aggregation and phase separation of monoclonal antibodies

“…Indeed, it has been shown previously that the Fab and Fc regions denature independently even within the context of the full-length molecule. 11,24 Although the IgG1-B Fab appeared to be thermodynamically more stable when isolated [by $ 2 C, see Table I and Fig. 3(B)], the magnitude of the difference did not invalidate our conclusions.…”

“…In contrast, the Fc fragment exhibits two minor cooperative transitions that partly overlap with the Fab melting transition and are due to the independent denaturation of C H 2 and C H 3 domains. 24 The extent of this overlap is known to be protein specific and highly dependent on the pH of the solution. 11 Generally, melting of the C H 2 domain occurs at lower temperatures compared to that of the C H 3 domain.…”

“…11 Generally, melting of the C H 2 domain occurs at lower temperatures compared to that of the C H 3 domain. 24 IgG2-A provides an example where the Fab is thermodynamically less stable and denatures within the same temperature range as the C H 2 domain. Unlike IgG2-A, the Fab portion of IgG1-A is more stable than the C H 2 and melts along with the C H 3 region.…”

“…36,38 In particular, the C H 2 domains of Fc are likely to be sufficiently destabilized or unfolded to initiate the aggregation process. 11,24 Aggregation of IgG1-A, IgG2-A, and IgG2-B was measured in 100 mM sodium acetate, pH 3.5. Unlike the high temperature and rigorous agitation experiments, all protein solutions remained free of visible protein precipitate for up to 48 h of static storage.…”

Thermodynamic and functional characterization of a stable IgG conformer obtained by renaturation from a partially structured low pH‐induced state

“…1). The lowest stability of the CH2 domain among IgG domains has been shown previously both at neutral and at acidic pH [14,15]. Therefore, it can be safely assumed that it is the CH2 domain which is partially 'unfolded' at pH 2.…”