Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy

Abstract

Protein aggregation generally results from association between hydrophobic regions of individual monomers. However, additional mechanisms arising from specific interactions, such as intermolecular disulfide bond formation, may also contribute to the process. The latter is proposed to be the initiating pathway for aggregation of immunoglobulin (IgG), which is essential for triggering its immune response. To test the veracity of this hypothesis, we have employed fluorescence correlation spectroscopy to measure the kinetics of aggregation of IgG in separate experiments either allowing or inhibiting disulfide formation. Fluorescence correlation spectroscopy measurements yielded a diffusion time (τ(D)) of ∼200 µsec for Rhodamine-labeled IgG, corresponding to a hydrodynamic radius (R(H)) of 56 Å for the IgG monomer. The aggregation kinetics of the protein was followed by monitoring the time evolution of τ(D) under conditions in which its cysteine residues were either free or blocked. In both cases, the progress curves confirmed that aggregation proceeded via the nucleation-dependent polymerization pathway. However, for aggregation in the presence of free cysteines, the lag times were shorter, and the aggregate sizes bigger, than their respective counterparts for aggregation in the presence of blocked cysteines. This result clearly demonstrates that formation of intermolecular disulfide bonds represents a preferred pathway in the aggregation process of IgG. Fluorescence spectroscopy showed that aggregates formed in experiments where disulfide formation was prevented denatured at lower concentration of guanidine hydrochloride than those obtained in experiments where the disulfides were free to form, indicating that intermolecular disulfide bridging is a valid pathway for IgG aggregation.

Unusual denaturation trajectory of bovine gamma globulin studied by fluorescence correlation spectroscopy

“…To account for both these effects, the change in t d of free RhB (40 nM) was measured as a function of GdnHCl concentration and the t d value for the protein at a particular GdnHCl concentration c was normalized by a multiplicative factor equal to the ratio (t d ) 0 /(t d ) c for RhB, where the subscript 0 refers to aqueous solution. 20,21,[41][42][43] Error bars of R H shown in Fig. 5-9 were calculated from the standard error of the mean of each t d from its five determinations for each sample.…”

Diffusion of Soluble Aggregates of THIOMABs and Bispecific Antibodies in Serum

“…30 Then, the average τ D of 2.52 ms for all Ab monomer samples yields an R H of 54 Å, which is in good agreement with other reported values of 55− 56 Å obtained by FCS. 31,32 The increase in τ D from Ab monomers to dimers was subtle but within the precision of the method, averaging just a 1.14-fold increase in apparent size. This similarity in diffusion properties for distinct oligomeric states is expected, because a given change in mass will cause a much smaller change in diffusion.…”

The Role of Geriatric Nutritional Risk Index in Predicting Postoperative Pulmonary Complications in Elderly Lung Cancer Patients Undergoing Surgical Resection

“…Globulin and LY% re ect the immune status of patients 22 . The cellular immunitymainly regulates immune function through T lymphocyte subsets 23 and in tumor surveillance, immunoglobulin contributes to target cell phagocytosis 24 .…”