Fibrillization Process of Human Amyloid-Beta Protein (1-40) under a Molecular Crowding Environment Mimicking the Interior of Living Cells Using Cell Debris.

Molecular crowding environments play a crucial role in understanding the mechanisms of biological reactions. Inside living cells, a diverse array of molecules coexists within a volume fraction ranging from 10% to 30% v/v. However, conventional spectroscopic methods often face difficulties in selectively observing the structures of particular proteins or membranes within such molecularly crowded environments due to the presence of high background signals. Therefore, it is crucial to establish in vitro measurement conditions that closely resemble the intracellular environment. Meanwhile, the neutron scattering method offers a significant advantage in selectively observing target biological components, even within crowded environments. Recently, we have demonstrated a novel scattering method capable of selectively detecting the structures of targeted proteins or membranes in a closely mimicking intracellular milieu achieved utilizing whole-cell contents (deuterated-cell debris). This method relies on the inverse contrast matching technique in neutron scattering. By employing this method, we successfully observed the fibrillization process of human amyloid beta-protein (Aß 1-40) under a molecular crowding environment (13.1% w/v cell debris, Aß/cell debris = ~1/25 w/w) that closely mimics the interior of living cells. Aß protein is well known as a major pathogenic component of Alzheimer's disease. The present results combining model simulation analyses clearly show that the intracellular environment facilitates the potential formation of even more intricate higher-order aggregates of Aß proteins than those previously reported.

Magnetic field effects on the structure and molecular behavior of pigeon iron-sulfur protein.

Pigeon iron-sulfur (Fe-S) cluster assembly 1 homolog (clISCA1) is a target protein for research into the biomagnetoreception mechanism, as the clCRY4/clISCA1 oligomer, a complex composed of the columnar clISCA1 oligomer and the magnetosensor candidate protein cryptochrome-4 (clCRY4) oligomer, tends to orient itself along weak magnetic fields, such as geomagnetic fields, under blue light. To obtain insight into the magnetic orientation mechanism of the clCRY4/clISCA1 oligomer, we inspected magnetic field effects on the structure and molecular behavior of clISCA1 by small angle X-ray scattering analysis. The results indicated that the clISCA1 protomer took the Fe-S cluster-bound globular form and unbound rod-like form. The globular clISCA1 protomer assembled to form columnar oligomers, which allowed for the binding of many Fe-S clusters at the interface between clISCA1 protomers. Moreover, the translational diffusion and the columnar oligomerization of clISCA1 were controlled by the external static magnetic field and Fe-S clusters bound to clISCA1. However, the columnar clISCA1 oligomer was not oriented along the external static magnetic field (~1 T) when clCRY4 was not bound to clISCA1. This result indicated that clCRY4 has a function to enhance the magnetic orientational property of clCRY4/clISCA1 oligomer.

Characterization of Localization, Ligand Binding, and pH-Dependent Conformational Changes of Two Chemosensory Proteins Expressed in the Antennae of the Japanese Carpenter Ant, Camponotus Japonicus.

Camponotus japonicus uses basiconic antennal sensilla (s. basiconica) to sense a colony-specific blend of species-specific cuticular hydrocarbons (CHCs). The inner portion of the s. basiconica is filled with sensillar lymph and chemosensory proteins (CSPs) presumed to transport CHCs to olfactory neuron receptors. Although 12 CSPs have been found in C. japonicus antennae, we focused on CjapCSP1 and CjapCSP13. The molecular basis of CSP1 function was explored by observation of its structure in solution at pH 4.0 and 7.0 through circular dichroism (CD) and X-ray solution scattering. Although the secondary structure did not vary with pH change, the radius of gyration (Rg) was larger by 5.3% (0.74 Å increase) at pH 4.0 than at pH 7.0. The dissociation constant (Kd) for CjapCSP1 measured with a fluorescent probe, 1-N-phenylnaphthylamine, was larger at pH 4.0 than at pH 7.0, suggesting that acidic pH triggers ligand dissociation. In contrast to CjapCSP1, the Rg of CjapCSP13 was slightly smaller at pH 4.0 than at pH 7.0. Western blotting and immunohistochemistry with protein-specific antisera revealed that both CjapCSP1 and CjapCSP13 are detected in the antennae, but differ in their specific internal localization. Binding to four compounds, including the ant CHC (z)-9-tricosene, was examined. Although both CjapCSP1 and CjapCSP13 bound to (z)-9-tricosene, CjapCSP13 bound with higher affinity than CjapCSP1 and showed different binding properties. CjapCSP1 and CjapCSP13 are synthesized by the same cells of the antenna, but function differently in CHC distribution due to differences in their localization and binding characteristics.

Short-Distance Intermolecular Correlations of Mono- and Disaccharides in Condensed Solutions: Bulky Character of Trehalose.

Organisms with tolerance to extreme environmental conditions (cryptobiosis) such as desiccation and freezing are known to accumulate stress proteins and/or sugars. Trehalose, a disaccharide, has received considerable attention in the context of cryptobiosis. It has already been shown to have the highest glass-transition temperature and different hydration properties from other mono- and disaccharides. In spite of the importance of understanding cryptobiosis by experimentally clarifying sugar-sugar interactions such as the clustering in concentrated sugar solutions, there is little direct experimental evidence of sugar solution structures formed by intermolecular interactions and/or correlation. Using a wide-angle X-ray scattering method with the real-space resolution from ∼3 to 120 Å, we clarified the characteristics of the structures of sugar solutions (glucose, fructose, mannose, sucrose, and trehalose), over a wide concentration range of 0.05-0.65 g/mL. At low concentrations, the second virial coefficients obtained indicated the repulsive intermolecular interactions for all sugars and also the differences among them depending on the type of sugar. In spite of the presence of such repulsive force, a short-range intermolecular correlation was found to appear at high concentrations for every sugar. The concentration dependence of the observed scattering data and p(r) functions clearly showed that trehalose prefers a more disordered arrangement in solution compared to other sugars, that is, bulky arrangement. The present findings will afford a new insight into the molecular mechanism of the protective functions of the sugars relevant to cryptobiosis, particularly that of trehalose.

Conformational selection of the intrinsically disordered plant stress protein COR15A in response to solution osmolarity - an X-ray and light scattering study.

The plant stress protein COR15A stabilizes chloroplast membranes during freezing. COR15A is an intrinsically disordered protein (IDP) in aqueous solution, but acquires an α-helical structure during dehydration or the increase of solution osmolarity. We have used small- and wide-angle X-ray scattering (SAXS/WAXS) combined with static and dynamic light scattering (SLS/DLS) to investigate the structural and hydrodynamic properties of COR15A in response to increasing solution osmolarity. Coarse-grained ensemble modelling allowed a structure-based interpretation of the SAXS data. Our results demonstrate that COR15A behaves as a biomacromolecule with polymer-like properties which strongly depend on solution osmolarity. Biomacromolecular self-assembly occurring at high solvent osmolarity is initiated by the occurrence of two specific structural subpopulations of the COR15A monomer. The osmolarity dependent structural selection mechanism is an elegant way for conformational regulation and assembly of COR15A. It highlights the importance of the polymer-like properties of IDPs for their associated biological function.

Observation of Protein and Lipid Membrane Structures in a Model Mimicking the Molecular-Crowding Environment of Cells Using Neutron Scattering and Cell Debris.

The interior of living cells is a molecular-crowding environment, where large quantities of various molecules coexist. Investigations into the nature of this environment are essential for an understanding of both the elaborate biological reactions and the maintenance of homeostasis occurring therein. The equilibrium states of biological macromolecular systems are affected by molecular-crowding environments unmatched by in vitro diluted environments; knowledge about crowding effects is still insufficient due to lack of relevant experimental studies. Recent developments in the techniques of in-cell NMR and large-scale molecular dynamics simulation have provided new insights into the structure and dynamics of biological molecules inside the cells. This study focused on a new experimental technique to directly observe the structure of a specific protein or membrane in condensed crowder solutions using neutron scattering. Deuterated whole-cell debris was used to reproduce an environment that more closely mimics the interior of living cells than models used previously. By the reduction of the background scattering from large amounts of cell debris, we successfully extracted structure information for both small globular protein and small unilamellar vesicle (SUV) from the concentrated cell-debris solution up to a weight ratio of 1:60 for protein/crowder and 1:40 for SUV/crowder.

Structure of Ultrafine Bubbles and Their Effects on Protein and Lipid Membrane Structures Studied by Small- and Wide-Angle X-ray Scattering.

Ultrafine bubbles (UFBs) are defined as small gas-filled bubbles with a diameter smaller than 1 µm. UFBs are stable for several weeks in aqueous solutions due to their small size. Although the mechanism of the stability of UFBs remains under intensive investigation, industrial applications of UFBs have recently arisen in various fields such as agricultural and fishery industries and medical therapy. The relevance of ions (protons and hydroxide anions) in UFB solutions has been discussed; however, the mechanism underlying the behavior of UFBs is still ambiguous and there is little direct evidence of the effect of UFBs on biological materials. This study deals with gaseous UFBs in aqueous solutions. Using small- and wide-angle X-ray scattering, we have investigated the structures of UFBs (air-UFBs, O2-UFBs, and N2-UFBs) and their effect on protein and lipid membrane structures. X-ray scattering and modeling data suggest that UFBs present a dynamic diffusive boundary (interface) due to the continuous release and absorption of gas. UFBs were found to not affect the structures of proteins at all hierarchal structure levels (from quaternary to tertiary, to internal, to secondary), whereas they did influence the packing and fluctuation of the hydrocarbon chains in the liposomes but not their shapes.

Restoration of Myoglobin Native Fold from Its Initial State of Amyloid Formation by Trehalose.

Organisms having tolerances against extreme environments produce and accumulate stress proteins and/or sugars in cells against the extreme environment such as high or low temperature, drying, and so forth. Sugars and/or polyols are known to prevent protein denaturation and enzyme deactivation. In particular, trehalose has received considerable attention because of its association with cryptobiosis and anhydrobiosis. This study focuses on the restoration of acid-denatured amyloid transition of myoglobin by trehalose. Myoglobin is known to proceed amyloidogenic reaction under denaturation conditions. We found that acid-denatured myoglobin at an initial process of amyloidogenic reaction (helix-to-sheet transition followed by oligomerization) at 25 °C was substantially restored to its native structure by trehalose. This action was prominent during the early stage of amyloid formation. Recent results showed that sugars are preferentially excluded from the protein surface to preserve its hydration shell and stabilize the protein structure against chemical and thermal denaturation. Therefore, the present results suggest that trehalose will restore the tightly bound water molecules around the hotspot (G-helix) of myoglobin on the amyloid transition by its intrinsic preservative action of the native hydration shell against denaturation. The present finding on the restorative action by trehalose could provide new insights into protein folding and amyloidosis.

Preferential Intercalation of Human Amyloid-ß Peptide into Interbilayer Region of Lipid-Raft Membrane in Macromolecular Crowding Environment.

This study focuses on the interaction of human amyloid ß-peptide (Aß) with a lipid-raft model membrane under macromolecular crowding conditions that mimic the intracellular environment. Aß is central to the development of Alzheimer's disease (AD) and has been studied extensively to determine the molecular mechanisms of Aß-induced cellular dysfunctions underlying the pathogenesis of AD. According to evidence from spectroscopic studies, ganglioside clusters are key to the fibrillization process of Aß. Gangliosides are a major component of glycosphingolipids and are acidic lipids of the central nervous system known to form so-called lipid rafts. In this study, the small unilamellar vesicle (SUV) membrane, composed of monosialogangliosides, cholesterol, and 1,2-dipalmitoyl- sn-glycero-3-phosphocholine, did not show any structural changes after the addition of Aß under noncrowding conditions. However, the addition of Aß under crowding conditions induced shape deformation and aggregation to SUV resulting in multilamellar stacking. The time evolution of the lamellar peak suggested the preferential cohesion or intercalation of the Aß peptide into the interbilayer region. This phenomenon was only observed at the gel (Lß) phase. These results suggest that an intracellular crowding environment promotes Aß-membrane interaction and a selective accumulation of Aß peptides into the interbilayer regions.

Sugar-Mediated Stabilization of Protein against Chemical or Thermal Denaturation.

The protective action of sugars against the denaturation of myoglobin was clarified by X-ray and neutron scattering methods. Different types of sugars such as disaccharides (trehalose, sucrose) and monosaccharides (glucose, fructose) were used. Experimental data and theoretical simulation based on three different solvation models (preferential solvation model, nonpreferential solvation model, and preferential exclusion (hydration) model) indicated that sugar molecules were preferentially or weakly excluded from the protein surface and preserved the native protein hydration shell. This trend was more evident for disaccharides. The preferential exclusion shifted gradually to the nonpreferential solvation at higher sugar concentrations. On the protective actions of the sugars against the guanidinium-chloride-mediated denaturation, all sugars, starting from the low concentration of 5% w/v, showed the protective trend toward the protein native structure, especially for the secondary structure. The thermal structural transition temperature of myoglobin was raised by about 4-5 °C, accompanied by amyloid formation, for all hierarchical structural levels. In particular, the oligomer formation of the amyloid aggregates was more suppressed. The above protective action was sugar-dependent. The present results clearly suggest that sugars intrinsically protect the native structure of proteins against chemical and thermal denaturation through the preservative action of the hydration shell.

Direct Evidence for the Effect of Glycerol on Protein Hydration and Thermal Structural Transition.

The mechanisms of protein stabilization by uncharged solutes, such as polyols and sugars, have been intensively studied with respect to the chemical thermodynamics of molecular crowding. In particular, many experimental and theoretical studies have been conducted to explain the mechanism of the protective action on protein structures by glycerol through the relationship between hydration and glycerol solvation on protein surfaces. We used wide-angle x-ray scattering (WAXS), small-angle neutron scattering, and theoretical scattering function simulation to quantitatively characterize the hydration and/or solvation shell of myoglobin in aqueous solutions of up to 75% v/v glycerol. At glycerol concentrations below ∼40% v/v, the preservation of the hydration shell was dominant, which was reasonably explained by the preferential exclusion of glycerol from the protein surface (preferential hydration). In contrast, at concentrations above 50% v/v, the partial penetration or replacement of glycerol into or with hydration-shell water (neutral solvation by glycerol) was gradually promoted. WAXS results quantitatively demonstrated the neutral solvation, in which the replacement of hydrated water by glycerol was proportional to the volume fraction of glycerol in the solvent multiplied by an exchange rate (ß ≤ 1). These phenomena were confirmed by small-angle neutron scattering measurements. The observed WAXS data covered the entire hierarchical structure of myoglobin, ranging from tertiary to secondary structures. We separately analyzed the effect of glycerol on the thermal stability of myoglobin at each hierarchical structural level. The thermal transition midpoint temperature at each hierarchical structural level was raised depending on the glycerol concentration, with enhanced transition cooperativeness between different hierarchical structural levels. The onset temperature of the helix-to-cross ß-sheet transition (the initial process of amyloid formation) was evidently elevated. However, oligomerization connected to fibril formation was suppressed, even at a low glycerol concentration.

Frozen State of Sephadex® Gels of Different Crosslink Density Analyzed by X-ray Computed Tomography and X-ray Diffraction.

Water in Sephadex® (crosslinked dextran) gels is known to indicate different freezing behavior which is dependent on the density of the crosslinks, and water in a Sephadex® G25 gel remains partially unfrozen during cooling and crystallizes during rewarming. The mechanism of anomalous ice crystallization during rewarming is still unclear. The objective of this study is to observe the ice grains that form in Sephadex® beads and to comprehend their frozen state with a focus on the ice crystallization during rewarming. Sephadex® beads containing 50 wt % water were prepared and used for the measurements. The observation of the ice grains was carried out by using synchrotron radiation-sourced X-ray CT (computed tomography). XRD (X-ray diffraction) analysis was also conducted to investigate the frozen state. As a result, ice grains that were larger than ~1 µm were hardly observed after the slow cooling of Sephadex® beads, except in the G25 beads. However, at the occurrence of ice crystallization during rewarming, ice grains that were larger than 10 µm appeared in the G25 beads. Using XRD, it was found that small incomplete ice crystals were formed in G25 beads and the presence of glassy water was indicated in the gel. In conclusion, the size and distribution of ice grains that formed in Sephadex® beads were different depending on the density of the crosslinks.

Effect of protein-encapsulation on thermal structural stability of liposome composed of glycosphingolipid/cholesterol/phospholipid.

We have studied the thermal structural stability of liposomes encapsulating proteins by using synchrotron radiation small- and wide-angle X-ray scattering (SR-SWAXS). Liposomes are known to be effective drug-delivery systems (DDSs) because they can reduce drug toxicity due to biodegradability and biocompatibility and can offer promising carriers of various types of drugs. However, in spite of numerous studies of liposomes, physicochemical characteristics of liposomes entrapping proteins are rarely known. The liposome studied is characterized by the lipid composition (mixture of acidic glycosphingolipid (ganglioside)/cholesterol/phospholipid). Gangliosides are one of the major constituents of so-called lipid rafts playing the role of a platform of cell-signaling. We have found that the encapsulation of proteins elevates the thermal transition temperature of the liposome membrane and suppresses the deformation of its shape. The present results suggest that not only membrane proteins, but also water-soluble proteins affect liposome stability through the revalence between osmotic pressure and membrane elasticity. In addition, we have found the presence of the size-effect depending on the molar content of gangliosides in the liposome, indicating the ability of ganglioside molecule controlling both the size and effective surface charge of the liposome. The present results would have significance from two different points of view. One is the confinement effect of proteins within a limited space like cell, and the other is a stability of a new type of DDS using gangliosides. Due to the intrinsic properties, gangliosides are expected to be promising agents for targeting and long-circulation properties of liposomal DDSs.

Structure of liposome encapsulating proteins characterized by X-ray scattering and shell-modeling.

Lipid liposomes are promising drug delivery systems because they have superior curative effects owing to their high adaptability to a living body. Lipid liposomes encapsulating proteins were constructed and the structures examined using synchrotron radiation small- and wide-angle X-ray scattering (SR-SWAXS). The liposomes were prepared by a sequential combination of natural swelling, ultrasonic dispersion, freeze-throw, extrusion and spin-filtration. The liposomes were composed of acidic glycosphingolipid (ganglioside), cholesterol and phospholipids. By using shell-modeling methods, the asymmetric bilayer structure of the liposome and the encapsulation efficiency of proteins were determined. As well as other analytical techniques, SR-SWAXS and shell-modeling methods are shown to be a powerful tool for characterizing in situ structures of lipid liposomes as an important candidate of drug delivery systems.

Kinetic asymmetry of subunit exchange of homooligomeric protein as revealed by deuteration-assisted small-angle neutron scattering.

We developed a novel, to our knowledge, technique for real-time monitoring of subunit exchange in homooligomeric proteins, using deuteration-assisted small-angle neutron scattering (SANS), and applied it to the tetradecamer of the proteasome α7 subunit. Isotopically normal and deuterated tetradecamers exhibited identical SANS profiles in 81% D(2)O solution. After mixing these solutions, the isotope sensitive SANS intensity in the low-q region gradually decreased, indicating subunit exchange, whereas the small-angle x-ray scattering profile remained unchanged confirming the structural integrity of the tetradecamer particles during the exchange. Kinetic analysis of zero-angle scattering intensity indicated that 1), only two of the 14 subunits were exchanged in each tetradecamer and 2), the exchange process involves at least two steps. This study underscores the usefulness of deuteration-assisted SANS, which can provide quantitative information not only on the molecular sizes and shapes of homooligomeric proteins, but also on their kinetic properties.

Polymer-dispersed bicontinuous cubic glycolipid nanoparticles.

We found that certain amphiphilic polymers such as PEO-PPO-PEO triblock copolymer (PL) can directly disperse a cubic glycolipid, 1-O-phytanyl-beta-D-xyloside (beta-XP), into bicontinuous cubic nanoparticles in water medium. The use of synchrotron small-angle X-ray diffraction (SSAXD) permitted the identification of the exact structure of these dispersed particles in the colloidal state. Dynamic light scattering method was used to obtain particle size distributions. The dispersion quality and the dispersion time can be improved by co-dissolving the lipid and the polymer in a common solvent. The mean volume diameter of these dispersed colloidal particles depends on the mixing time and polymer concentration. About 5 wt % (0.18 mol %) of polymer to lipid weight was found to be sufficient to produce stable colloidal dispersions. At this polymer content and at 3 h of stirring time, the mean volume diameter of cubic colloidal particles was found to be 1.0 microm. Increase of dispersion time to 6 h reduced the colloidal particle size from 1.0 microm to 660 nm. At 3 h of mixing time, the increase of polymer content, from approximately 5 to approximately 10 wt %, reduced the particle mean diameter from 1.0 microm to 675 nm. Irrespective of these dispersion times and polymer contents, the dispersed colloidal particles exhibit predominately the Pn3m cubic phase structure, the same as that of a beta-XP-water binary mixture, although a weak coexistence of Im3m cubic phase is identified in these colloidal particles. This coexistence is found to have the characteristics of a Bonnet relation, which forms convincing evidence for the infinite periodic minimal surface descriptions (IPMS). Considering the biotechnological significance, the preparation of these colloidal dispersions was carried out in a phosphate-buffered saline (PBS) system. These cubic colloidal dispersions exhibited good stability and the cubic phase structure remained intact in the PBS system.

Glycolipid based cubic nanoparticles: preparation and structural aspects.

Kinetically stable cubic colloidal particle dispersion was produced from a glycolipid using a novel preparation strategy based on the dialysis principle. The use of synchrotron small-angle X-ray diffraction (SSAXD) permitted the identification of exact structure of these dispersed particles in the colloidal state. Dynamic light scattering methods were used to obtain size and size distributions. A glycoside, 1-O-phytanyl-beta-D-xyloside (beta-XP), that exhibits Pn3m cubic phase in an excess aqueous medium, was used as the lipid material. The dialysis technique includes controlled stirring action both inside and outside of the dialysis membrane tube. Initially, a mixed micellar system composed of beta-XP, n-octyl-beta-D-glucopyranoside (beta-OG) and a triblock copolymer, Pluronic F127 (PL) was prepared in the aqueous medium. About 10 wt.% of PL to lipid weight was found to be sufficient to produce stable colloidal dispersions. The mean volume diameter of these colloidal particles was found to be in the range of 0.85 +/-0.05 microm. The cubic phase structure of these colloidal particles is greatly depended on the final beta-OG concentration level in the system. Coexistence of Im3m and Pn3m cubic structures has been identified in these colloidal particles. This coexistence has the characteristics of Bonnet relation, which forms a compelling case for the infinite periodic minimal surface (IPMS) descriptions. These colloidal particles could restore pure Pn3m phase structure, but a longer dialysis time was needed. This work, in general, will open up new possibilities for membrane protein reconstitution and other relevant biological applications using colloidal cubic lipid particles.

Hierarchical map of protein unfolding and refolding at thermal equilibrium revealed by wide-angle X-ray scattering.

Hierarchical features of the thermal unfolding-refolding structural transition of hen egg white lysozyme (HEWL) have been studied in the temperature range from 13 to 84 degrees C by using high-resolution wide-angle X-ray scattering (WAXS) measurements at a third-generation synchrotron source. We have gathered high-statistic WAXS data of the reversible unfolding-refolding process of HEWL in the q range from approximately 0.05 to approximately 3 A(-1) [q = (4pi/lambda) sin(theta/2), where theta is the scattering angle and lambda the wavelength]. This measured q range corresponds to the spatial distance from approximately 2 to approximately 125 A, which covers all hierarchical structures of a small globular protein such as HEWL, namely, tertiary, domain, and secondary structures. Because of this, we have found that the pH dependence of the thermal structural transition of HEWL is well characterized by the various hierarchical levels and the transition concurrence among them. In this report, we present a new hierarchical map depiction of unfolding-refolding transitions. Using scattering with various ranges of q values, we determine the molar ratio of native-like protein structure defined by the data in each range, thus producing a map of the amount of native-like structure as a function of the hierarchical level or resolution. This map can visualize a detailed feature of the unfolding-refolding transition of a protein depending on various structural hierarchical levels; however, the exact meaning of the map will await sharpening by additional works.

An assay of ganglioside using fluorescence image analysis on a thin-layer chromatography plate.

A new and simple fluorometric method for determine quantities of gangliosides ranging from pico- to nanomoles is reported. Spraying hydrochloric acid followed by a heating treatment, sugars (glucose, galactose, fucose, N-acetylgalactosamine, N-acetylglucosamine, N-acetylneuraminic acid (sialic acid)), gangliosides (G(M1), G(D1a), G(T1b)), and asialoganglioside (asialoG(M1)) on thin-layer chromatography plates produced fluorescence under 365-nm UV light. This fluorescence production of each sample was greatly dependent on the heating temperature. As sialic acid fluoresced readily at lower temperature (approximately 90 degrees C), we were able to distinguish sialic acid easily from other sugars tested. To determine gangliosides based on this sialic acid fluorescence, calibration curves for gangliosides were obtained by high-performance thin-layer chromatography and the image-analyzing system equipped with a CCD camera. The observed fluorescence images were analyzed using image-analyzing software packages and the determined calibration curves for ganglioside-bound sialic acids were reproducible and showed a high linearity in a wide range from 47 pmol to 4.5 nmol. Since the fluorescence from sialic acid is easily measurable on TLC plates and is sensitive over a wide range of sample concentration, the present method is applicable for quantitative determination of gangliosides.