proteins STRUCTURE O FUNCTION O BIOINFORMATICS Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy Moupriya Nag, Kallol Bera, and Soumen Basak* Chemical Sciences Division, Saha Institute of Nuclear Physics, Bidhannagar, Kolkata 700064, India ABSTRACT Protein aggregation generally results from association between hydrophobic regions of individual monomers. However, addi- tional 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. Fluores- cence correlation spectroscopy measurements yielded a diffusion time (s D ) of ~200 msec for Rhodamine-labeled IgG, corre- sponding to a hydrodynamic radius (R H ) of 56 A ˚ for the IgG monomer. The aggregation kinetics of the protein was followed by monitoring the time evolution of s 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 intermo- lecular 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 disul- fide bridging is a valid pathway for IgG aggregation. Proteins 2015; 83:169–177. V C 2014 Wiley Periodicals, Inc. Key words: immunoglobulin; fluorescence correlation spectroscopy; disulfide; protein aggregation; nucleation dependent polymerization. INTRODUCTION Blood serum comprises of approximately 7% protein, two-thirds of which is in the albumin fraction and one third in the globulin fraction. Globulins are a class of globular proteins (molecular weight  150 kDa) catego- rized as alpha, beta, and gamma globulins according to increasing size. Some of the gamma globulins are immu- nologically active and are called immunoglobulins (IgG’s). 1–3 The structure of IgG comprises of four poly- peptide chains: two identical light chains (molecular weight 22–23 kDa) and two identical heavy chains (molecular weight 50–70 kDa). 4 These four polypeptide chains are arranged in the characteristic “Y”-shape formed by three distinct globular domains that have sep- arate existence and biological functions. The amino acid sequence in some domains varies from one IgG molecule to another, and hence they are known as variable domains (F ab ). The remaining domains have approxi- mately similar sequence within groups of IgG’s and are therefore termed constant domain (F C ). This structural bifurcation into a variable (F ab ) and a constant part (F C ) Additional Supporting Information may be found in the online version of this article. Grant sponsor: CSIR, Government of India; Grant sponsor: The BARD Project at Saha Institute. *Correspondence to: S. Basak, Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064, India. E-mail: soumen.basak@saha.ac.in or soumenbasak08@gmail.com Received 1 April 2014; Revised 21 October 2014; Accepted 28 October 2014 Published online 5 November 2014 in Wiley Online Library (wileyonlinelibrary. com). DOI: 10.1002/prot.24715 V V C 2014 WILEY PERIODICALS, INC. PROTEINS 169