Cysteines in ß-lactoglobulin affects its interfacial adsorption and protein film stabilization.

HYPOTHESIS: Disulfide bonds in proteins are strong chemical bonds forming the secondary and tertiary structure like in the dairy protein ß-lactoglobulin. We hypothesize that the partial or complete removal of disulfide bonds affects the structural rearrangement of proteins caused by intra- and intermolecular interactions that in turn define the interfacial activity of proteins at oil/water interfaces. The experimental and numerical investigations contribute to the mechanistic understanding of the structure-function relationship, especially for the interfacial adsorption behavior of proteins. EXPERIMENTAL AND NUMERICAL: Systematically, the 5 cysteines of ß-lactoglobulin were recombinantly exchanged by alanine. First, the protein structure of the variants in bulk was analyzed with Fourier-transform-infrared-spectroscopy and molecular dynamic simulations. Second, the structural changes after adsorption to the interface have been also analyzed by molecular dynamic simulations. The adsorption behavior was investigated by pendant drop analysis and the interfacial film properties by dilatational rheology. FINDINGS: The structural flexibility of ß-lactoglobulin with no cysteines encourages its unfolding at the interface, and accelerates the interfacial protein film formation that results in more visco-elastic films in comparison to the reference.

Regioselective analysis of heat-induced conformational changes of ß-lactoglobulin by quantitative liquid chromatography-mass spectrometry analysis of chemical labeling kinetics.

ß-Lactoglobulin is a main allergen in cow's milk; its allergenicity is strongly impacted by processing. To understand heat-induced epitope-specific effects, the present study analyzed regiospecific conformational changes of heated native ß-lactoglobulin variant A (BLG-A). Complementary fluorescence spectroscopy methods indicated two denaturation phases comprising minor sequential conformational changes (25-75 °C) and complete transitions (80-90 °C). Regioselective conformational changes of BLG-A in the native state (25 °C), sequential (70 °C) and complete transition (90 °C) were determined by quantitative liquid chromatography-mass spectrometry analysis of chemical labeling kinetics covering 14 lysine residues and the N-terminus. Conformational changes in two phases were observed for N-terminus, K8 (both N-terminal chain), K60 (ß-sheet C), K75 (ß-sheet D), K77 (DE loop), K83 (ß-sheet E), K100 and K101 (FG loop). The residues K14 (ß-sheet A1), K47 (ß-sheet B), K69, K70 (both ß-sheet D), and K91 (ß-sheet F) were not involved in conformational changes.

Carboxymethyl chitosan coating infused with linalool-loaded molten globular ß-Lactoglobulin nanoparticles for extended preservation of fresh-cut apples.

This investigation employed molten globule state ß-lactoglobulin nanoparticles (MG-BLGNPs) for encapsulating linalool (LN) combined with carboxymethyl chitosan (CMC) coating to enhance the shelf-life of fresh-cut apples. The effect of different MG structures on the encapsulation efficiency of BLGNPs and the properties of coating was studied. Structural characterization and molecular simulation showed structural differences between heat-induced MG state (70-BLGNPs, heated at 70 °C for 1 h) and sodium dodecyl sulfate-co-heat-induced MG state (SDS/70-BLGNPs, treated with 0.192 mg/mL SDS for 10 min, then heated at 70 °C for 1 h), with the latter being more unfolded. LN self-assembles into MG-BLGNPs, among the generated particles, SDS/70-BLG@LN exhibits stronger binding effect and higher LN loading capacity. Integration of MG-BLG@LN into CMC enhanced coating's mechanical properties and adhesion to fresh-cut apples. The SDS/70-BLG@LN/CMC coating showed superior preservation on fresh-cut apples during storage, reducing enzymatic browning, membrane lipid oxidation, and microbial growth while maintaining hardness and overall quality.

Exploration of dynamic interaction between ß-lactoglobulin and casein micelles during UHT milk process.

The protein aggregation induced by UHT treatment shortens the shelf life of UHT milk. However, the mechanism of ß-Lg induced casein micelle aggregation remains unclear. Herein, the dynamic interaction between ß-Lg and casein micelles during UHT processing was investigated by experimental techniques and molecular dynamics simulations. Results showed that ß-Lg decreased the stability of casein micelles, increased their size and zeta potential. Raman and FTIR spectra analysis suggested that hydrogen and disulfide bonds facilitated their interaction. Cryo-TEM showed that the formation of the casein micelle/ß-Lg complex involved rigid binding, flexible linking, and severe cross-linking aggregation during UHT processing. SAXS and MST demonstrated ß-Lg bound to κ-casein on micelle surfaces with a dissociation constant (Kd) of 3.84 ± 1.14 µm. Molecular docking and dynamic simulations identified the interacting amino acid residues and clarified that electrostatic and van der Waals forces drove the interaction. UHT treatment increased hydrogen bonds and decreased total binding energy. The non-covalent binding promoted the formation of disulfide bonds between ß-Lg and casein micelles under heat treatment. Ultimately, it was concluded that non-covalent interaction and disulfide bonding resulted in casein micelle/ß-Lg aggregates. These findings provided scientific insights into protein aggregation in UHT milk.

ß-Lactoglobulin-based amorphous solid dispersions: A graphical review on the state-of-the-art.

Proteins have recently caught attention as potential excipients for amorphous solid dispersions (ASDs) to improve oral bioavailability of poorly water-soluble drugs. Notably, the studies have highlighted whey protein isolates, particularly ß-lactoglobulin (BLG), as promising candidates in amorphous stabilization, dissolution and solubility enhancement, achieving drug loadings of 50 wt% and higher. Consequently, investigations into the mechanisms underlying the solid-state stabilization of amorphous drugs and the enhancement of drug solubility in solution have been conducted. This graphical review provides a comprehensive overview of recent findings concerning BLG-based ASDs. Firstly, the dissolution performance of BLG-based ASDs is compared to more traditional polymer-based ASDs. Secondly, the drug loading onto BLG and the resulting amorphous stabilization mechanisms is summarized. Thirdly, interactions between BLG and drug molecules in solution are described as the mechanisms governing the improvement of drug solubility. Lastly, we outline the impact of the spray drying process on the secondary structure of BLG, and the resulting differences in amorphous stabilization and drug dissolution performance between α-helix-rich and ß-sheet-rich BLG-based ASDs.

Purification of α-lactalbumin and ß-lactoglobulin from cow milk.

Whey, a valuable byproduct of dairy processing, contains essential proteins like ß-lactoglobulin (ßLG) and α-lactalbumin (αLA), making it a focus of research for its nutritional benefits. Various techniques, including chromatography and membrane filtration, are employed for protein extraction, often requiring multiple purification steps. One approach that has gained prominence for the purification and concentration of proteins, including those present in whey, is the use of polyethylene glycol (PEG) in aqueous two-phase systems. Our study simplifies this process by using PEG alone for whey protein purification. This approach yielded impressive results, achieving 92 % purity for ßLG and 90 % for αLA. These findings underscore the effectiveness of PEG-based purification in isolating whey proteins with high purity.

Exploration of Novel Plasmin Inhibitor from ß-Lactoglobulin for Enhancing the Storage Stability of UHT Milk.

Plasmin-induced protein hydrolysis significantly compromises the stability of ultrahigh-temperature (UHT) milk. ß-Lactoglobulin (ß-Lg) was observed to inhibit plasmin activity, suggesting that there were active sites as plasmin inhibitors in ß-Lg. Herein, plasmin inhibitory peptides were explored from ß-Lg using experimental and computational techniques. The results revealed that increased denaturation of ß-Lg enhanced its affinity for plasmin, leading to a stronger inhibition of plasmin activity. Molecular dynamics simulations indicated that electrostatic and van der Waals forces were the primary binding forces in the ß-Lg/plasmin complex. Denatured ß-Lg increased hydrogen bonding and reduced the binding energy with plasmin. The sites of plasmin bound to ß-Lg were His624, Asp667, and Ser762. Four plasmin inhibitory peptides, QTMKGLDI, EKTKIPAV, TDYKKYLL, and CLVRTPEV, were identified from ß-Lg based on binding sites. These peptides effectively inhibited plasmin activity and enhanced the UHT milk stability. This study provided new insights into the development of novel plasmin inhibitors to improve the stability of UHT milk.

Effects of high pressure processing on structural changes, aggregation, and binding mechanisms of ß-Lactoglobulin with typical polyphenols.

The binding capacity of ß-Lactoglobulin (BLG) is crucial for delivering polyphenols, influenced by structural changes. High pressure processing (HPP) has the potential to modify BLG's structure and aggregation, but its specific impact on BLG-polyphenol interactions is uncertain. This study used circular dichroism spectroscopy and molecular dynamics simulations to reveal HPP-induced structural changes in BLG, supported by particle size analysis indicating aggregation. Seven structurally diverse polyphenols (quercetin-QR, hesperetin-HSP, dihydromyricetin-DHM, gallic acid-GA, (-)-epicatechin-EC, resveratrol-RES, and secoisolariciresinol diglucoside-SDG) were investigated to comprehensively analyze their binding patterns using fluorescence spectroscopy and molecular docking. HPP reduced BLG's ordered structure and increased its aggregation. Binding affinities peaked at 400 MPa for DHM, QR, HSP, GA, and RES, while SDG and EC exhibited maximum affinities at atmospheric pressure and 600 MPa, respectively. Elevated pressures enhanced BLG-polyphenol interactions, particularly at residues 44GLU and 160CYS, with van der Waals forces dominating the binding free energy.

Allergenicity assessment of ß-lactoglobulin ferulic acid-glucose conjugates.

We prepared the ß-lactoglobulin (BLG)-ferulic acid (FA)-glucose (Glu) conjugates by alkaline method and Maillard reaction to assess the allergenicity. FA and Glu can form a ternary covalent conjugate with BLG, as evidenced by the shortening of SEC retention time, upward migration of SDS-PAGE protein bands, considerable decrease in free amino and sulfhydryl content, and changes in multistructure. BLG-Glu-FA conjugates weakly bound to immunoglobulin E in allergic sera was weak, reduced interleukin 4 and tumor necrosis factor α levels in RBL-2H3 cells and histamin and interleukin 6 secretion levels in KU812 cells, and inhibited the nuclear factor-κB signaling pathway. In vivo experiments showed that the conjugates regulated T-cell homeostasis in mouse splenic and mesenteric lymphocytes and attenuated splenic and duodenal immune injury. Therefore, the conjugates of BLG with FA combined with Glu altered the epitope structure and exhibited low allergenicity.

Identification of proteolytic bacteria from Yunnan fermented foods and their use to reduce the allergenicity of ß-lactoglobulin.

Beta-lactoglobulin (ß-LG) is considered to be the major allergenic protein in milk. Lactic acid bacteria (LAB) possess a protein hydrolysis system that holds great promise for hydrolyzing ß-LG and reducing its allergenicity. Therefore, this study aimed to screen LAB with ß-LG hydrolysis activity from Yunnan traditional fermented foods. The results showed that Pediococcus pentosaceus C1001, Pediococcus acidilactici E1601-1, and Lactobacillus paracasei E1601-2, could effectively hydrolyze ß-LG and further reduce its sensitization (more than 40%). All 3 lactic acid bacteria hydrolyzed ß-LG allergenic fragments V41-K60 and L149-I162. Moreover, they encode a variety of genes related to proteolysis, such as aminopeptidase pepC and pepN, proline peptidase pepIP and endopeptidase pepO, and L. paracasei E1601-2 contains extracellular protease coding gene prtP. And they encode a variety of genes associated with hydrolyzed proteins. The 3 strains screened in this study can be used to develop hypoallergenic dairy products.

CRISPR-mediated editing of ß-lactoglobulin (BLG) gene in buffalo.

Milk is a good source of nutrition but is also a source of allergenic proteins such as α-lactalbumin, ß-lactoglobulin (BLG), casein, and immunoglobulins. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas technology has the potential to edit any gene, including milk allergens. Previously, CRISPR/Cas has been successfully employed in dairy cows and goats, but buffaloes remain unexplored for any milk trait. In this study, we utilized the CRISPR/Cas9 system to edit the major milk allergen BLG gene in buffaloes. First, the editing efficiency of designed sgRNAs was tested in fibroblast cells using the T7E assay and Sanger sequencing. The most effective sgRNA was selected to generate clonal lines of BLG-edited cells. Analysis of 15 single-cell clones, through TA cloning and Sanger sequencing, revealed that 7 clones exhibited bi-allelic (-/-) heterozygous, bi-allelic (-/-) homozygous, and mono-allelic (-/+) disruptions in BLG. Bioinformatics prediction analysis confirmed that non-multiple-of-3 edited nucleotide cell clones have frame shifts and early truncation of BLG protein, while multiple-of-3 edited nucleotides resulted in slightly disoriented protein structures. Somatic cell nuclear transfer (SCNT) method was used to produce blastocyst-stage embryos that have similar developmental rates and quality with wild-type embryos. This study demonstrated the successful bi-allelic editing (-/-) of BLG in buffalo cells through CRISPR/Cas, followed by the production of BLG-edited blastocyst stage embryos using SCNT. With CRISPR and SCNT methods described herein, our long-term goal is to generate gene-edited buffaloes with BLG-free milk.

Gut Microbiome-Serum Metabolism Revealed the Allergenicity of Ferulic Acid Combined with Glucose-Modified ß-Lactoglobulin.

A novel strategy combining ferulic acid and glucose was proposed to reduce ß-lactoglobulin (BLG) allergenicity and investigate whether the reduction in allergenicity was associated with gut microbiome and serum metabolism. As a result, the multistructure of BLG changed, and the modified BLG decreased significantly the contents of IgE, IgG, IgG1, and mMCP-1 in serum, improved the diversity and structural composition of gut microbiota, and increased the content of short-chain fatty acids (SCFAs) in allergic mice. Meanwhile, allergic mice induced by BLG affected arachidonic acid, tryptophan, and other metabolic pathways in serum, the modified BLG inhibited the production of metabolites in arachidonic acid metabolism pathway and significantly increased tryptophan metabolites, and this contribution helps in reducing BLG allergenicity. Overall, reduced allergenicity of BLG after ferulic acid was combined with glucose modification by regulating gut microbiota, the metabolic pathways of arachidonic acid and tryptophan. The results may offer new thoughts alleviating the allergy risk of allergenic proteins.

Molecular Dynamics Insights into the Aggregation Behavior of N-Terminal ß-Lactoglobulin Peptides.

ß-lactoglobulin (BLG) forms amyloid-like aggregates at high temperatures, low pH, and low ionic strengths. At a pH below 2, BLG undergoes hydrolysis into peptides, with N-terminal peptides 1-33 and 1-52 being prone to fibrillization, forming amyloid-like fibrils. Due to their good mechanical properties, BLG amyloids demonstrate great potential for diverse applications, including biosensors, nanocomposites, and catalysts. Consequently, further studies are essential to comprehensively understand the factors governing the formation of BLG amyloid-like morphologies. In this study, all-atom molecular dynamics simulations were employed to explore the aggregation of N-terminal 1-33 and 1-52 BLG peptides under conditions of pH 2 and at 10 mM NaCl concentration. The simulations revealed that the peptides spontaneously assembled into aggregates of varying sizes. The aggregation process was enabled by the low charge of peptides and the presence of hydrophobic residues within them. As the peptides associated into aggregates, there was a concurrent increase in ß-sheet structures and the establishment of hydrogen bonds, enhancing the stability of the aggregates. Notably, on average, 1-33 peptides formed larger aggregates compared to their 1-52 counterparts, while the latter exhibited a slightly higher content of ß-sheets and higher cluster orderliness. The applied approach facilitated insights into the early stages of amyloid-like aggregation and molecular-level insight into the formation of ß-sheets, which serve as nucleation points for further fibril growth.

Electrostatic interactions mediated defibrillation of ß-lactoglobulin fibrils using Keggin Polyoxometalates.

The whey protein ß-lactoglobulin (ßLG) forms fibrils similar to the amyloid fibrils in the neurodegenerative diseases due to its higher predisposition of ß-sheets. This study shed light on the understanding different inorganic Keggin polyoxometalates (POMs) interaction with the protein ßLG fibrils. POMs such as Phosphomolybdic acid (PMA), silicomolybdic acid (SMA), tungstosilicic acid (TSA), and phosphotungstic acid (PTA) were used due to their inherent higher anionic charges. The interaction studies were monitored with fluorescence spectra and Thioflavin T assay for both the ßLG monomers and the fibrils initially to elucidate the binding ability of the POMs. The binding of POMs and ßLG is also demonstrated by molecular docking studies. Zeta potential studies showed the electrostatic mediated higher interactions of the POMs with the protein fibrils. Isothermal titration calorimetry (ITC) studies showed that the molybdenum containing POMs have higher affinity to the protein fibrils than the tungsten. This study could help understanding formation of food grade protein fibrils which have profound importance in food industries.

Exploring the effect of protein secondary structure on the solid state and physical stability of protein-based amorphous solid dispersions.

Amorphous solid dispersions (ASDs) using proteins as carriers have emerged as a promising strategy for stabilizing amorphous drug molecules. Proteins possess diverse three-dimensional structures that significantly influence their own properties and may also impact the properties of ASDs. We prepared ß-lactoglobulin (BLG) with different contents of ß-sheet and α-helical secondary structures by initially dissolving BLG in different mixed solvents, containing different ratios of water, methanol/ethanol, and acetic acid, followed by spray drying of the solutions. Our findings revealed that an increase in α-helical content resulted in a decrease in the glass transition temperature (Tg) of the protein. Subsequently, we utilized the corresponding mixed solvents to dissolve both BLG and the model drug celecoxib (CEL), allowing the preparation of ASDs containing either ß-sheet-rich or α-helix/random coil-rich BLG. Using spray drying, we successfully developed BLG-based ASDs with drug loadings ranging from 10 wt% to 90 wt%. At drug loadings below 40 wt%, samples prepared using both methods exhibited single-phase ASDs. However, heterogeneous systems formed when the drug loading exceeded 40 wt%. At higher drug loadings, physical stability assessments demonstrated that the α-helix/random coil-rich BLG structure exerted a more pronounced stabilizing effect on the drug-rich phase compared to the ß-sheet-rich BLG. Overall, our results highlight the importance of considering protein secondary structure in the design of ASDs.

Structural and tribological studies on the interaction of porcine gastric mucin with non- and cationic-modified ß-lactoglobulins.

ß-lactoglobulin (BLG) is the major whey protein with negative charges at neutral pH in aqueous media. Thus, the interaction with mucins, the major polyanionic component of mucus, is very weak due to the electrostatic repulsion between them. The present study postulates that cationization of BLG molecules may reverse the interaction characteristics between BLG and mucin from repulsive to associative. To this end, cationic-modified BLGs were prepared by grafting positively charged ethylenediamine (EDA) moieties into the negatively charged carboxyl groups on the aspartic and glutamic acid residues and compared with non-modified BLG upon mixing with porcine gastric mucin (PGM). To characterize the structural and conformational features of PGM, non/cationized BLGs, and their mixtures, various spectroscopic approaches, including zeta potential, dynamic light scattering (DLS), and circular dichroism (CD) spectroscopy were employed. Importantly, we have taken surface adsorption with optical waveguide lightmode spectroscopy (OWLS), and tribological properties with pin-on-disk tribometry at the sliding interface as the key approaches to determine the interaction nature between them as mixing PGM with polycations can lead to synergistic lubrication at the nonpolar substrate in neutral aqueous media as a result of an electrostatic association. All the spectroscopic studies and a substantial improvement in lubricity collectively supported a tenacious and associative interaction between PGM and cationized BLGs, but not between PGM and non-modified BLG. This study demonstrates a unique and successful approach to intensify the interaction between BLG and mucins, which is meaningful for a broad range of disciplines, including food science, macromolecular interactions, and biolubrication etc.

The mechanism of epigallocatechin-3-gallate inhibiting the antigenicity of ß-lactoglobulin under pH 6.2, 7.4 and 8.2: Multi-spectroscopy and molecular simulation methods.

The antigenicity of ß-lactoglobulin (ß-LG) can be influenced by pH values and reduced by epigallocatechin-3-gallate (EGCG). However, a detailed mechanism concerning EGCG decreasing the antigenicity of ß-LG at different pH levels lacks clarity. Here, we explore the inhibition mechanism of EGCG on the antigenicity of ß-LG at pH 6.2, 7.4 and 8.2 using enzyme-linked immunosorbent assay, multi-spectroscopy, mass spectrometry and molecular simulations. The results of Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) elucidate that the noncovalent binding of EGCG with ß-LG induces variations in the secondary structure and conformations of ß-LG. Moreover, EGCG inhibits the antigenicity of ß-LG the most at pH 7.4 (98.30 %), followed by pH 6.2 (73.18 %) and pH 8.2 (36.24 %). The inhibitory difference is attributed to the disparity in the number of epitopes involved in the interacting regions of EGCG and ß-LG. Our findings suggest that manipulating pH conditions may enhance the effectiveness of antigenic inhibitors, with the potential for further application in the food industry.

Enhanced in vitro bioavailability of resveratrol-loaded emulsion stabilized by ß-lactoglobulin-catechin with excellent antioxidant activity.

The study aimed to fabricate ß-Lactoglobulin-catechin (ß-La-Ca) conjugates as a natural designed antioxidant emulsifier to improve the physicochemical stability of resveratrol emulsion delivery system. Fourier transform infrared (FT-IR) and fluorescence spectroscopy analysis confirmed the formation of conjugates using free radical grafting. The antioxidant ability of emulsion was evaluated by DPPH scavenging activities and ORAC experiments. The emulsion stabilized by ß-La-Ca conjugates exhibited strong antioxidant activity with ORAC value of 2541.39 ± 29.58 µmol TE/g, which was significantly higher than that by ß-Lactoglobulin alone with 387.96 ± 23.45 µmol TE/g or their mixture with 948.23 ± 32.77 µmol TE/g. During the whole simulated gastrointestinal digestion, emulsion stabilized by ß-La-Ca conjugates exhibited excellent oxidative stability that the lipid was mainly digested in the small intestine. This behavior attributed to the greater stability of resveratrol to chemical transformation leading to a higher overall bioavailability in vivo. These results suggested that the ß-La-Ca conjugates could be used to fabricate the emulsion-based delivery system to improve the oxidative stability and bioavailability of chemically labile hydrophobic bioactive compounds.

Architecture of ß-lactoglobulin coating modulates bioinspired alginate dialdehyde-gelatine/polydopamine scaffolds for subchondral bone regeneration.

In this study, we developed polydopamine (PDA)-functionalized alginate dialdehyde-gelatine (ADA-GEL) scaffolds for subchondral bone regeneration. These polymeric scaffolds were then coated with ß-Lactoglobulin (ß-LG) at concentrations of 1 mg/ml and 2 mg/ml. Morphological analysis indicated a homogeneous coating of the ß-LG layer on the surface of network-like scaffolds. The ß-LG-coated scaffolds exhibited improved swelling capacity as a function of the ß-LG concentration. Compared to ADA-GEL/PDA scaffolds, the ß-LG-coated scaffolds demonstrated delayed degradation and enhanced biomineralization. Here, a lower concentration of ß-LG showed long-lasting stability and superior biomimetic hydroxyapatite mineralization. According to the theoretical findings, the single-state, representing the low concentration of ß-LG, exhibited a homogeneous distribution on the surface of the PDA, while the dimer-state (high concentration) displayed a high likelihood of uncontrolled interactions. ß-LG-coated ADA-GEL/PDA scaffolds with a lower concentration of ß-LG provided a biocompatible substrate that supported adhesion, proliferation, and alkaline phosphatase (ALP) secretion of sheep bone marrow mesenchymal stem cells, as well as increased expression of osteopontin (SPP1) and collagen type 1 (COL1A1) in human osteoblasts. These findings indicate the potential of protein-coated scaffolds for subchondral bone tissue regeneration. STATEMENT OF SIGNIFICANCE: This study addresses a crucial aspect of osteochondral defect repair, emphasizing the pivotal role of subchondral bone regeneration. The development of polydopamine-functionalized alginate dialdehyde-gelatine (ADA-GEL) scaffolds, coated with ß-Lactoglobulin (ß-LG), represents a novel approach to potentially enhance subchondral bone repair. ß-LG, a milk protein rich in essential amino acids and bioactive peptides, is investigated for its potential to promote subchondral bone regeneration. This research explores computationally and experimentally the influence of protein concentration on the ordered or irregular deposition, unravelling the interplay between coating structure, scaffold properties, and in-vitro performance. This work contributes to advancing ordered protein coating strategies for subchondral bone regeneration, providing a biocompatible solution with potential implications for supporting subsequent cartilage repair.

The Interaction between Anesthetic Isoflurane and Model-Biomembrane Monolayer Using Simultaneous Quartz Crystal Microbalance (QCM) and Quartz Crystal Impedance (QCI) Methods.

The interaction between anesthetic Isoflurane (Iso) and model-biomembrane on the water surface has been investigated using quartz crystal microbalance (QCM) and quartz crystal impedance (QCI) methods. The model-biomembranes used were dipalmitoyl phosphatidyl choline (DPPC), DPPC-palmitic acid (PA) mixture (DPPC:PA = 8:2), DPPC-Alamethicin (Al) mixture (DPPC:Al = 39:1), and DPPC-ß-Lactoglobulin (ßLG) mixture (DPPC:ßLG = 139:1) monolayers, respectively. The quartz crystal oscillator (QCO) was attached horizontally to each monolayer, and QCM and QCI measurements were performed simultaneously. It was found that Iso hydrate physisorbed on each monolayer/water interface from QCM and changed those interfacial viscosities from QCI. With an increase in Iso concentration, pure DPPC, DPPC-PA mixed, and DPPC-Al mixed monolayers showed a two-step process of Iso hydrate on both physisorption and viscosity, whereas it was a one-step for the DPPC-ßLG mixed monolayer. The viscosity change in the DPPC-ßLG mixed monolayer with the physisorption of Iso hydrate was much larger than that of other monolayers, in spite of the one-step process. From these results, the action mechanism of anesthetics and their relevance to the expression of anesthesia were discussed, based on the "release of interfacial hydrated water" hypothesis on the membrane/water interface.