Exposure Of Hydrophobic Regions Research Articles

Modulation of textural properties in microwave treated gluten-free quinoa sponge cake by alkaline amino acids

Lysine (Lys) and Arginine (Arg) (0.6%) was added to the quinoa flour to improve the textural quality and physical properties of gluten-free sponge cake treated with different heating methods. The gluten-free sponge cake fortified Lys and Arg increased cell density and surface area fraction, compared to water bath (WB) treatment, with the microwave (MW) treatment further enhancing these effects. Furthermore, MW combined Lys treatment showed the best texture characteristics of sponge cake. The water distribution determined by 1H NMR presented that water molecules are bound by the protein network structure. FTIR spectrum proved MW combined with Lys promoted the transition from disordered structure to ordered structure of protein in quinoa sponge cake, which enhanced the expansion of protein molecules and the exposure of hydrophobic regions, thus promoting protein aggregation. SDS-PAGE revealed when alkaline amino acids were present, they can regulate the ordered aggregation of protein molecules under MW alternating electric field, leading to enhanced intermolecular interactions. Finally, microstructure evaluations by SEM demonstrated MW combined with alkaline amino acids formed a stable three-dimensional gel network structure, thus improving the quality of gluten-free quinoa sponge cake. Therefore, the study findings pointed to the feasibility of using MW to improve the physical and textual quality of quinoa gluten-free food.

High pressure-assisted enzymatic hydrolysis promotes the release of a bi-functional peptide from cowhide gelatin with dipeptidyl peptidase IV (DPP-IV) inhibitory and antioxidant activities

The process of generating functional peptides from cowhide gelatin is challenged by inefficient enzymatic hydrolysis. In this study, the researchers attempted to enhance the hydrolysis and potential functional properties of the peptides by subjecting the cowhide gelatin to high-pressure treatment (200, 300, and 400 MPa) for 20 min, followed by enzymatic hydrolysis. The highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, 2,2′ azinobis(3 ethylbenzothiazoline 6 sulfonic acid) (ABTS) radical scavenging activity, and DPP-IV inhibitory activity of the hydrolysate were obtained at 200 MPa, accompanied with an increase in the content of hydrophobic, acidic, and basic amino acids (P < 0.05). Correspondingly, the high-pressure pretreatment (200 MPa) reduced the thermal stability, particle size, and morphological integrity of cowhide gelatin, with a corresponding increase in the exposure of hydrophobic regions. Altogether, these results indicated that appropriate high-pressure-assisted enzymatic hydrolysis reinforced the release of bi-functional peptides by modifying the structure of cowhide gelatin.

Effects of ultrasonic treatment on ovomucin: Structure, functional properties and bioactivity

The effects of ultrasonic treatment on the structure, functional properties and bioactivity of Ovomucin (OVM) were investigated in this study. Ultrasonic treatment could significantly enhance OVM solubility without destroying protein molecules. The secondary structure changes, including β-sheet reduction and random coil increase, indicate more disorder in OVM structure. After ultrasonic treatment, the OVM molecule was unfolded partially, resulting in the exposure of hydrophobic regions. The changes in OVM molecules led to an increase in intrinsic fluorescence and surface hydrophobicity. By detecting the particle size of protein solution, it was confirmed that ultrasonic treatment disassembled the OVM aggregations causing a smaller particle size. Field emission scanning electron microscopy (FE-SEM) images showed that ultrasonic cavitation significantly reduced the tendency of OVM to form stacked lamellar structure. Those changes in structure resulted in the improvement of foaming, emulsification and antioxidant capacity of OVM. Meanwhile, the detection results of ELISA showed that ultrasonic treatment did not change the biological activity of OVM. These results suggested that the relatively gentle ultrasound treatment could be utilized as a potential approach to modify OVM for property improvement.

Changes in structure, rheological property and antioxidant activity of soy protein isolate fibrils by ultrasound pretreatment and EGCG

Impacts of ultrasound pretreatment on the formation of soy protein fibrils, interaction of soy protein fibrils and EGCG, rheological property and antioxidant activity of the complex, and bioaccessibility of EGCG were investigated in this study. Fourier-transform infrared (FTIR) spectroscopy, fluorescence spectroscopy, and surface hydrophobicity (H0) indicated that ultrasound caused the unfolding of soy protein structure and exposure of hydrophobic regions. Compared with the un-treated soy protein fibrils, there were greater Thioflavin T fluorescence intensity and β-sheet content in the ultrasound-treated protein fibrils. These results indicated that ultrasound pretreatment could be used for modifying soy protein to improve its fibrils formation. FTIR spectroscopy, fluorescence spectroscopy, X-ray diffraction, and H0 showed that protein fibrils mainly interacted with EGCG through hydrogen bonds and hydrophobic interactions. The stability, viscosity, and antioxidant activity of soy protein fibrils treated with ultrasound-assisted EGCG (U–F-EGCG) were greater than those of other samples. EGCG drove the formation of protein fibrils-EGCG hydrogels. In vitro bioaccessibility of EGCG was significantly improved after its formation of complex with protein fibrils. Hence, the soy protein fibril hydrogels system can extend the application as the delivery carriers of bioactive compounds.

Deltamethrin modulates the native structure of Hen Egg White Lysozyme and induces its aggregation at physiological pH

Deltamethrin, a type II pyrethroid pesticide was initially considered as safe for human use. Recent studies have reported several pathophysiological effects of deltamethrin on human and non-human species. However, its effect on structure and function of protein leading to progressive neurodegeneration is poorly understood. In present study, we investigated the interaction of deltamethrin with Hen Egg White Lysozyme (HEWL) at physiological pH and tried to understand the effect of pesticide on structure and function of protein. Employing different biophysical techniques, we shown that deltamethrin induces in vitro aggregation of HEWL in concentration dependent manner. Interaction of pesticide with different amino acids, followed by exposure of hydrophobic regions was driving force of aggregation process. Apart from modulating the hydrophobic domain, deltamethrin is observed to reduce α-helical and promote β-sheet content of lysozyme, eventually converting the globular protein into ThT sensitive amyloid fibrils and amorphous aggregates. Our study also indicate that deltamethrin induced aggregation reduces the catalytic activity of lysozyme.

Inhibition of tau aggregation and associated cytotoxicity on neuron-like cells by calycosin

In this study, the in vitro assembly of tau and anti-amyloidogenic properties of one naturally occurring phytoestrogen, calycosin, was investigated by spectroscopic techniques including ThT and ANS fluorescence, CD, Congo red absorbance as well as TEM analysis. Afterwards the cytotoxicity of different amyloid species against SH-SY5Y cells was evaluated by MTT assay. Fluorescence spectroscopic studies revealed that calycosin exerts its anti-amyloidogenic effects through increasing the lag time and reducing the apparent growth rate constant (kapp), the amount of fibrillation, and the exposure of hydrophobic regions. Congo red absorbance and CD studies indicated that calycosin prevented the formation of tau aggregate species and β-sheets structures, respectively. TEM analysis also determined the capacity of calycosin to inhibit tau fibrillogenesis through formation of large amorphous aggregates. Furthermore, cellular assays disclosed that calycosin mitigated the cell mortality, LDH release, ROS level, and expression of Bax, Bcl-2, and Caspase-3 in both mRNA and protein levels induced by tau amyloid fibrils. In conclusion, this data may suggest that calycosin can prevent tau amyloid fibrillation and the associated cytotoxicity, mainly due to its effects on formation of lower content of oligomeric and fibrillar aggregates with lower solvent-exposed hydrophobic patches compared to those produced in the absence of calycosin.

Hydrophobicity and aggregation, but not glycation, are key determinants for uptake of thermally processed β-lactoglobulin by THP-1 macrophages

The aim of this study is to investigate the immunological relevance of modifications of food protein structure due to thermal processing. We investigated the uptake of β-lactoglobulin, treated with 3 different processing methods, by THP-1 macrophages: wet heating (60 °C in solution) and high- or low-temperature (130 °C or 50 °C, respectively) dry heating, combined with either of 8 types of saccharides or without saccharide. The processing method that was applied significantly affected the uptake of BLG by THP-1 macrophages, while the type of saccharide only had an influence in high-temperature dry heated samples. A set of physicochemical parameters of processed samples was determined, to determine the samples' molecular weight, hydrophobicity, amyloid-like structure, surface charge and secondary structure. Analysis of protein structure alterations indicated the uptake to be linked to the wet heating processing method and percentage of α-helix structure, amyloid-like structures, polymers, and hydrophobicity. We hypothesize that both amyloid-like structures and molecular weight were related to the increased hydrophobicity and therefore postulate that the exposure of hydrophobic regions is the leading physicochemical characteristic for the observed uptake of wet heated BLG samples by THP-1 macrophages. This work demonstrates how differential thermal processing of foods, through protein modification, can have an impact on its interaction with the immune system.

Characterization of Denatured States and Reversible Unfolding of Sensory Rhodopsin II

Our understanding on the folding of membrane proteins lags behind that of soluble proteins due to challenges posed by the exposure of hydrophobic regions during in vitro chemical denaturation and refolding experiments. While different folding models are accepted for soluble proteins, only the two-stage model and the long-range interactions model have been proposed so far for helical membrane proteins. To address our knowledge gap on how different membrane proteins traverse their folding pathways, we have systematically investigated the structural features of SDS-denatured states and the kinetics for reversible unfolding of sensory rhodopsin II (pSRII), a retinal-binding photophobic receptor from Natronomonas pharaonis. pSRII is difficult to denature, and only SDS can dislodge the retinal chromophore without rapid aggregation. Even in 30% SDS (0.998 ΧSDS), pSRII retains the equivalent of six out of seven transmembrane helices, while the retinal-binding pocket is disrupted, with transmembrane residues becoming more solvent exposed. Folding of pSRII from an SDS-denatured state harboring a covalently bound retinal chromophore shows deviations from an apparent two-state behavior. SDS denaturation to form the sensory opsin apo-protein is reversible. We report pSRII as a new model protein which is suitable for membrane protein folding studies and has a unique folding mechanism that differs from those of bacteriorhodopsin and bovine rhodopsin.

Proton pump inhibitors drastically modify triosephosphate isomerase from Giardia lamblia at functional and structural levels, providing molecular leads in the design of new antigiardiasic drugs

BackgroundProton pump inhibitors (PPIs) are extensively used in clinical practice because of their effectiveness and safety. Omeprazole is one of the best-selling drugs worldwide and, with other PPIs, has been proposed to be potential drugs for the treatment of several diseases. We demonstrated that omeprazole shows cytotoxic effects in Giardia and concomitantly inactivates giardial triosephosphate isomerase (GlTIM). Therefore, we evaluated the efficiency of commercially available PPIs to inactivate this enzyme. MethodsWe assayed the effect of PPIs on the GlTIM WT, single Cys mutants, and the human counterpart, following enzyme activity, thermal stability, exposure of hydrophobic regions, and susceptibility to limited proteolysis. ResultsPPIs efficiently inactivated GlTIM; however, rabeprazole was the best inactivating drug and was nearly ten times more effective. The mechanism of inactivation by PPIs was through the modification of the Cys 222 residue. Moreover, there are important changes at the structural level, the thermal stability of inactivated-GlTIM was drastically diminished and the structural rigidity was lost, as observed by the exposure of hydrophobic regions and their susceptibility to limited proteolysis. ConclusionsOur results demonstrate that rabeprazole is the most potent PPI for GlTIM inactivation and that all PPIs tested have substantial abilities to alter GITIM at the structural level, causing serious damage.General significance.This is the first report demonstrating the effectiveness of commercial PPIs on a glycolytic parasitic enzyme, with structural features well known. This study is a step forward in the use and understanding the implicated mechanisms of new antigiardiasic drugs safe in humans.

Evidence for a Molten Globule State in Cicer α-Galactosidase Induced by pH, Temperature, and Guanidine Hydrochloride

Physiologically as well as industrially, α-galactosidases are very important enzymes, but very little is known about the stability and folding aspect of enzyme. In the present study, we have investigated the temperature, pH, and guanidine hydrochloride (GuHCl) induced unfolding of Cicer α-galactosidase using circular dichroism and fluorescence spectroscopy. Strong negative ellipticities at 208, 215, and 222 nm indicate the presence of both α and β structures in Cicer α-galactosidase and showed that its secondary structure belongs to α + β class of proteins with 31 % α-helicity. For Cicer α-galactosidase the emission maximum was found to be 345 nm which suggests that tryptophan residues are less exposed to solvent. However, at pH 2.0, protein showed blue-shift. This state of protein lacked activity but it retained significant secondary structure. Enhanced binding of ANS at pH 2.0 indicated significant unfolding and exposure of hydrophobic regions. The unfolded state of Cicer α-galactosidase showed a red-shift of 15 nm with a concomitant decrease in the fluorescence intensity. The enzyme maintained its native structure and full activity up to 40 °C; however, above this temperature, denaturation was observed.

Exploring the unfolding mechanism of γ-glutamyltranspeptidases: The case of the thermophilic enzyme from Geobacillus thermodenitrificans

γ-glutamyltranspeptidases (γ-GTs) are ubiquitous enzymes that catalyze the hydrolysis of γ-glutamyl bonds in glutathione and glutamine and the transfer of the released γ-glutamyl group to amino acids or short peptides. These enzymes are generally synthesized as precursor proteins, which undergo an intra-molecular autocatalytic cleavage yielding a large and a small subunit. In this study, circular dichroism and intrinsic fluorescence measurements have been used to investigate the structural features and the temperature- and guanidinium hydrochloride (GdnHCl)-induced unfolding of the mature form of the γ-GT from Geobacillus thermodenitrificans (GthGT) and that of its T353A mutant, which represents a mimic of the precursor protein. Data indicate that a) the mutant and the mature GthGT have a different secondary structure content and a slightly different exposure of hydrophobic regions, b) the thermal unfolding processes of both GthGT forms occur through a three-state model, characterized by a stable intermediate species, whereas chemical denaturations proceed through a single transition, c) both GthGT forms exhibit remarkable stability against temperature, but they do not display a strong resistance to the denaturing action of GdnHCl. These findings suggest that electrostatic interactions significantly contribute to the protein stability and that both the precursor and the mature form of GthGT assume compact and stable conformations to resist to the extreme temperatures where G. thermodenidrificans lives. Owing to its thermostability and unique catalytic properties, GthGT is an excellent candidate to be used as a glutaminase in food industry.

Correlation of membrane binding and hydrophobicity to the chaperone-like activity of PDC-109, the major protein of bovine seminal plasma.

The major protein of bovine seminal plasma, PDC-109 binds to choline phospholipids present on the sperm plasma membrane upon ejaculation and plays a crucial role in the subsequent events leading to fertilization. PDC-109 also shares significant similarities with small heat shock proteins and exhibits chaperone-like activity (CLA). Although the polydisperse nature of this protein has been shown to be important for its CLA, knowledge of other factors responsible for such an activity is scarce. Since surface exposure of hydrophobic residues is known to be an important factor which modulates the CLA of chaperone proteins, in the present study we have probed the surface hydrophobicity of PDC-109 using bisANS and ANS. Further, effect of phospholipids on the structure and chaperone-like activity of PDC-109 was studied. Presence of DMPC was found to increase the CLA of PDC-109 significantly, which could be due to the considerable exposure of hydrophobic regions on the lipid-protein recombinants, which can interact productively with the nonnative structures of target proteins, resulting in their protection. However, inclusion of DMPG instead of DMPC did not significantly alter the CLA of PDC-109, which could be due to the lower specificity of PDC-109 for DMPG as compared to DMPC. Cholesterol incorporation into DMPC membranes led to a decrease in the CLA of PDC-109-lipid recombinants, which could be attributed to reduced accessibility of hydrophobic surfaces to the substrate protein(s). These results underscore the relevance of phospholipid binding and hydrophobicity to the chaperone-like activity of PDC-109.

Novel therapeutic strategies for the treatment of protein-misfolding diseases

Most proteins in the cell adopt a compact, globular fold that determines their stability and function. Partial protein unfolding under conditions of cellular stress results in the exposure of hydrophobic regions normally buried in the interior of the native structure. Interactions involving the exposed hydrophobic surfaces of misfolded protein conformers lead to the formation of toxic aggregates, including oligomers, protofibrils and amyloid fibrils. A significant number of human disorders (e.g. Alzheimer disease, Parkinson disease, Huntington disease, amyotrophic lateral sclerosis and type II diabetes) are characterised by protein misfolding and aggregation. Over the past five years, outstanding progress has been made in the development of therapeutic strategies targeting these diseases. Three promising approaches include: (1) inhibiting protein aggregation with peptides or small molecules identified via structure-based drug design or high-throughput screening; (2) interfering with post-translational modifications that stimulate protein misfolding and aggregation; and (3) upregulating molecular chaperones or aggregate-clearance mechanisms. Ultimately, drug combinations that capitalise on more than one therapeutic strategy will constitute the most effective treatment for patients with these devastating illnesses.

Combined spectroscopic and calorimetric characterisation of rubredoxin reversible thermal transition

Rubredoxins are small iron proteins containing the simplest type of iron-sulphur centre, consisting of an iron atom coordinated by the thiol groups of four cysteines. Here we report studies on the conformational stability of a new type of rubredoxin from the hyperthermophile Methanocaldococcus jannaschii, having an atypical metal site geometry resulting from a modified iron-binding motif. Absorption and fluorescence spectroscopies were used in combination with differential scanning calorimetry to probe different aspects of the thermal unfolding transition: iron site degradation (absorption at 380 nm), tertiary structure unfolding (Trp emission), exposure of hydrophobic regions (1-anilinonaphalene-8-sulphonate fluorescence enhancement) and iron release. Thermal denaturation was found to be irreversible and caused by decomposition of the metal centre. The protein is hyperstable and between pH 4 and 10 it is only thermally denatured in the presence of a strong chemical denaturant. The study of the heating rate dependence of the melting temperature allowed us to determine the reaction equilibrium thermodynamic parameters. At pH 2 the protein is destabilised owing to the absence of salt bridges and it has a T(m) of 65 degrees C. In these conditions, there is excellent agreement between the parameters determined by the different spectroscopic methods and calorimetry. The highest stability was found to be at pH 8, and a detailed study of the heating rate dependence in the presence of guanidine thiocyanate in this condition allowed the determination of a reversible T(m) of 118 degrees C.

Proteasomal Degradation of Mutant Superoxide Dismutases Linked to Amyotrophic Lateral Sclerosis

Mutations in copper-zinc superoxide dismutase cause the neurodegenerative disease amyotrophic lateral sclerosis. Many of the mutant proteins have increased turnover in vivo and decreased thermal stability. Here we show that purified, metal-free superoxide dismutases are degraded in vitro by purified 20 S proteasome in the absence of ATP and without ubiquitinylation, whereas their metal-bound counterparts are not. The rate of degradation by the proteasome varied among the mutants studied, and the rate correlated with the in vivo half-life. The monomeric forms of both mutant and wild-type superoxide dismutase are particularly susceptible to degradation by the proteasome. Exposure of hydrophobic regions as a consequence of decreased thermal stability may allow the proteasome to recognize these molecules as non-native.

Characterization of a partially folded intermediate of stem bromelain at low pH.

Equilibrium studies on the acid included denaturation of stem bromelain (EC 3.4.22.32) were performed by CD spectroscopy, fluorescence emission spectroscopy and binding of the hydrophobic dye, 1-anilino 8-naphthalene sulfonic acid (ANS). At pH 2.0, stem bromelain lacks a well defined tertiary structure as seen by fluorescence and near-UV CD spectra. Far-UV CD spectra show retention of some native like secondary structure at pH 2.0. The mean residue ellipticities at 208 nm plotted against pH showed a transition around pH 4.5 with loss of secondary structure leading to the formation of an acid-unfolded state. With further decrease in pH, this unfolded state regains most of its secondary structure. At pH 2.0, stem bromelain exists as a partially folded intermediate containing about 42.2% of the native state secondary structure Enhanced binding of ANS was observed in this state compared to the native folded state at neutral pH or completely unfolded state in the presence of 6 m GdnHCl indicating the exposure of hydrophobic regions on the protein molecule. Acrylamide quenching of the intrinsic tryptophan residues in the protein molecule showed that at pH 2.0 the protein is in an unfolded conformation with more tryptophan residues exposed to the solvent as compared to the native conformation at neutral pH. Interestingly, stem bromelain at pH 0.8 exhibits some characteristics of a molten globule, such as an enhanced ability to bind the fluorescent probe as well as considerable retention of secondary structure. All the above data taken together suggest the existence of a partially folded intermediate state under low pH conditions.

Pressure denaturation and aggregation of beta-lactoglobulin studied by intrinsic fluorescence depolarization, Rayleigh scattering, radiationless energy transfer and hydrophobic fluoroprobing.

beta-Lactoglobulin (beta-lg) in aqueous solution under pressure showed a marked depolarization of intrinsic fluorescence assigned to a gradually increased rotational diffusion of tryptophyl moieties in pressure-unfolded states. The corresponding change in anisotropy provided a new and more accurate method for determining denaturation volume which, for beta-lg in neutral aqueous solution with ionic strength 0.16 (NaCl) at 25 degrees C, was delta V degree = -73 (SE 3) ml mol-1, corresponding to half denaturation at 123 MPa. The pressure unfolding led to exposure of hydrophobic regions to the protein-water interface that could be probed by fluorescence intensity of a beta-lg-1-anilinonaphthalene-8-sulphonic acid (ANS) complex with 1:1 stoichiometry, as determined by Job's method of continuous variation. The unfolding of beta-lg impaired the binding capacity of the inner calyx, with a reduction in binding capacity of 50% at 50 MPa, as shown by decreasing cis-parinaric acid fluorescence, decreasing anisotropy and decreasing radiationless energy transfer from tryptophans to this probe with increasing pressure. The pressure-induced reversible exposure of hydrophobic groups to the protein-water interface may, at least partly, explain the initial aggregation reactions, evident from increased Rayleigh scattering from approximately 50 MPa, prior to irreversible pressure-induced gel formation of beta-lg. Using results from this and previous studies, we propose a three step pressure denaturation model for beta-lg for neutral solution at ambient temperature, including an initial pressure-melted state (up to 50 MPa) with partial collapse of the inner calyx and solvent exposure of the free thiol group, followed by a reversible denaturation with exposure of hydrophobic regions (half denaturation at 123 MPa) and with irreversible denaturation with thiol-disulphide exchange becoming increasingly important at higher pressures. Effects of pressure on beta-lg, as measured by fluorescence depolarization, were found for the reversible denaturation steps to be similar to the effects of chemical denaturants but different with respect to shift in ANS emission maxima.

Interactions of lipoprotein lipase with the active-site inhibitor tetrahydrolipstatin (Orlistat).

Lipoprotein lipase (LPL) was rapidly inactivated by low concentrations of the active-site inhibitor tetrahydrolipstatin (THL). The presence of amphiphils (e.g. long-chain fatty acids) or of lipid/water interfaces (lipid emulsions) was required for inhibition to occur. Apolipoprotein CII increased the maximal inactivation rate constant by 1.8-fold in the presence of an emulsion of long-chain triacylglycerols, but had no effect in the presence of an emulsion of tributyrylglycerol. The fully inhibited enzyme had a ratio of THL/LPL of nearly 2, indicating that both subunits of the LPL homo-dimer bound THL. The THL-LPL complex was stable below pH 7.5. At higher pH reactivation occurred indicating that THL was slowly turned over by the enzyme. The apparent reactivation rate constant was increased about threefold by the presence of lipid/water interfaces. Sucrose density gradient centrifugation revealed that THL induces tetramerisation of LPL. This aggregation was reversible on reactivation of the inhibited enzyme. Binding to heparin was not affected by THL. In contrast, binding to lipid droplets and to lipoproteins was increased, indicating exposure of hydrophobic regions in the inhibited LPL. It is suggested that THL induces local conformational changes in LPL, which may involve opening of the putative surface lid structure which covers the active-site.

6] Monitoring protein conformational changes during membrane fusion

This chapter discusses on the various procedures for detection of protein conformational change and exposure of hydrophobic regions during membrane fusion. These procedures can be roughly classified into biophysical, biochemical, and immunological methods. Procedures for the detection of the penetration of hydrophobic segments of proteins into bilayer membranes occurring through conformational changes are also outlined. Protein conformational changes and membrane insertion are usually detected under conditions that trigger membrane fusion. Protein conformational changes are reflected most directly by circular dichroism (CD) spectra: CD spectra in the far- and near-ultraviolet regions are sensitive to changes in peptide backbone structure and the conformation around aromatic groups. Ultraviolet spectra in the near-ultraviolet regions are also sensitive to conformational changes around aromatic amino acid residues: red and blue spectral shifts are observed on perturbation of tyrosine and/or tryptophan residues by a solvent, and on their exposure to a more polar environment.

Receptor-mediated interaction of ricin with the lipid bilayer of ganglioside G M1-liposomes

The interaction of ricin with ganglioside G M1 or glycoprotein containing liposomes was investigated. At neutral pH, ricin bound to galactose moieties on the surface of the liposomes to form ricin-liposome complexes, but did not associate with their lipid bilayers. When these ricin-liposome complexes were exposed to a pH below 5, ricin bound to G M1-liposomes became associated with the lipid bilayer, whereas ricin bound to glycoprotein-liposomes (containing human erythrocyte Band 3) was only rarely associated. Association of ricin with the lipid bilayer of G M1-liposomes did not occur in the presence of lactose, which inhibits the binding of ricin to ganglioside G M1. Using a hydrophobic probe, 8-anilino-1-naphthalene sulfonic acid (ANS), it was revealed that an acidity below pH 5 resulted in exposure of hydrophobic regions on the ricin molecule. These results strongly suggest that association of ricin with the lipid bilayer of G M1-liposomes at acidic pH is mediated by the binding of ricin to ganglioside G M1 at neutral pH and occurs through interaction between the exposed hydrophobic region on the ricin molecule and the lipid bilayer of G M1-liposomes at low pH.