Increased selectivity of sodium deoxycholate to around Tryptophan213 in bovine serum albumin upon micellization as revealed by singular value decomposition for excitation emission matrix

Subunit vaccines are commonly used against influenza viruses. The epitope of the viral antigen is administered alongside immunostimulant adjuvant. Adjuvants are used to increase the efficacy of some drugs and are responsible for inhibiting the progression of virus-induced symptoms by stimulating the production of antibodies against the virus. There are many adjuvants for influenza vaccines. They include Freund’s adjuvant and MF59, also known as emulsion adjuvants, because they contain surfactants used to solubilize membrane proteins. In some cases, emulsion adjuvants may denature the antigen proteins.

Surfactant-induced denaturation of antigens could be beneficial to immunostimulatory effects. This suggests the importance of understanding a substance’s effect on the antigen’s steric structure when looking for adjuvant candidates. Additionally, knowledge of the conformational changes of antigens could provide more insights into the action mechanism of adjuvants, whose mechanisms are still unclear. For example, knowledge of the adjuvant mechanism, which converts non-antigenic proteins into antigens, is important in understanding the pathogenesis of allergies.

Bile salts (sodium deoxycholate, NaDC), is a promising adjuvant candidate owing to their favorable gelation properties. Like surfactant Quillaja saponaria, NaDC has a similar steroid skeleton and intestinal absorption-promoting effect and is also a potential candidate for oral vaccine components. Additionally, NaDC is a valuable membrane permeation enhancer for oral vaccines. The 3D structure and antigenicity of Bovine serum albumin (BSA) have been extensively studied. BSA comprises three distinct drug-binding domains: domain I containing tryptophan (Trp) 134 and domain II containing Trp213, from which the BSA intrinsic fluorescence is commonly derived from. Therefore, separating the fluorescence of these two Trps would provide more insights into the conformational behavior of BSA.

Herein, Mr. Yuya Kurosawa, Assistant Professor Yuta Otsuka and Professor Satoru Goto from Tokyo University of Science investigated the effects of NaDC on the conformation of globular proteins. In a similar study, nuclear magnetic resonance, thermograms, fluorescence spectra and circular dichroism spectra were measured to elucidate the structural changes in the proteins. Specifically, excitation–emission matrice was adopted here owing to its ability to scan the excitation wavelength and reveal the local environment of the amino acid residues in the protein. It is also suitable for characterizing multiple fluorescent molecules.

In their approach, BSA was chosen as the model antigen. The NaDC concentration was set to cross the critical micelle concentration due to the expected differences in the effects of surfactants on proteins depending on the aggregate. A ratio of the micelle concentration was used as a unit to establish the relationship between the surfactant concentration and the critical micelle concentration. The elements in the fluorescence spectra were separated through singular value decomposition (SVD). Their work is currently published in the journal, Colloids and Surfaces B: Biointerfaces.

The authors observed that the Trp residues of BSA and the fluorescence energy of NaDC were in a three-way relationship, with each element in the fluorescence spectra being effectively separated via SVD. Besides having a large effect on the microenvironment around Trp213, NaDC also enhanced the selective confirmation of Trp213 in the BSA. These results were confirmed by experiments using ketoprofen specific domain II and warfarin specific domain I, where NaDC induced selectivity in domain II that mostly consisted of Trp213. Additionally, micellization improved the selectivity of NaDC to Trp213 while selectively was highly influenced by the aggregation state of the NaDC.

In summary, the research team successfully separated the fluorescent components, NaDC, and Trp residues in BSA by applying SVD to excitation–emission matrices. NaDC induced selective and localized conformational changes in BSA. Furthermore, this method is applicable to the dilution of protein solutions to provide more insights into the interaction between surfactants and proteins without the need for NMR. In a joint statement the authors told Advances in Engineering, that their findings would contribute to the formulation of NaDC as a membrane permeation enhancer or adjuvant in oral vaccines.