Study of Binding between Protein A and Immunoglobulin G Using a Surface Tension Probe

Abstract

Molecular interactions and binding are one of the most important and fundamental properties in the study of biochemical and biomedical systems. The understanding of such interactions and binding among biomolecules forms the basis for the design and processing of many biotechnological applications, such as bioseparation and immunoadsorption. In this study, we present a novel method to probe molecular interactions and binding based on surface tension measurement. This method complements conventional techniques, which are largely based on optical, spectroscopic, fluorescence polarization, chromatographic or atomic force microscopy measurements, by being definite in determining molecular binding ratio and flexible in sample preparation. Both dynamic and equilibrium (or quasi-equilibrium) information on molecular binding can be obtained through dynamic and equilibrium surface tension measurements. For an important pair of biological ligand and ligate, Protein A and immunoglobulin G (IgG), the existence of molecular interactions and the binding ratio of 1:2 have been determined unequivocally with the proposed surface tension method. These results are confirmed/supported by a mass balance calculation and spectrophotometry experiment. In addition, adsorption isotherms for Protein A and IgG separately at the air/water interface have been established with the dynamic surface tension measurements. The results show that the Langmuir isotherm equation can describe the adsorption data satisfactorily for both Protein A and IgG solutions.

Study of protein–protein binding reaction by whole‐column fluorescence‐imaged CIEF

“…It is known that molecular weights of protein A and human IgG are 42 and 150 kDa [11], respectively. Molecular weight of FITC-protein A was calculated to be about 43.4 kDa from the weight ratio of FITC to protein A in the FITC-protein A.…”

“…Therefore, studies of the interactions between a ligand and target protein or protein and protein are important to understand insights of the biochemical and biomedical processes. The methods employed in the determination of ligand-protein or protein-protein interactions include fluorescence correlation spectroscopy [4,5], fluorescence polarization [6], X-ray optical method [7], chromatographic method [8], radioactive method [9], atomic force microscopy [10], surface tension probe method [11], CE [12,13], and so on.…”

“…Further studies [27,28] stipulated a 1:1 molar-binding ratio. Recently, surface tension probe method indicated that the molar-binding ratio of protein A and IgG was 1:2 [11]. It is thus necessary and interesting to use an independent experiment to determine the binding ratio.…”

Separation of antigens and antibodies by immunoaffinity chromatography

“…Between the C H1 and C H 2 is the hinge region. An IgG molecule can be divided into two parts functionally: Fragment antigen-binding (Fab) fragment, which is the antigen-binding site, and Fragment crystallizable (Fc) fragment, which is the protein A-binding site (Yang et al, 2003). (b) The Staphylococcal protein A shown here is comprised with five homologous IgG-binding domains (E, D, A-C), which have high affnity with the side chains of His435 and Tyr436 of Fc1, and a cell-wall attaching structure (XM) (Hober et al, 2007).…”

Modeling of adsorption dynamics at air–liquid interfaces using statistical rate theory (SRT)

“…The water used for the solution preparation was purified by an Ultra-Pure water system from Millipore Co., with a resistivity of 18.2 M . The details of the experimental procedure can be found elsewhere [32]. The parameters involved in the Langmuir isotherm for C 10 E 8 were obtained from Ref.…”