Mutual recognition between polymerized liposomes. IV. Polysaccharide-lectin system.

As a model of cell-cell recognition processes, the association processes of a polysaccharide (mannan)-carrying liposome with a lectin (Concanavalin A, Con A)-carrying polymerized liposome were followed by turbidimetry. The association process was strongly inhibited by the addition of a low molecular weight sugar, methyl-alpha-D-mannopyranoside, which shows that the association between the liposomes is due to the specific interaction between Con A and mannan. The association rate constant obtained was much smaller than the theoretical value for a diffusion-controlled binary association process. This implies that the association rate of liposomes is limited by the recognition between complementary ligands bound on the liposome surfaces. Another reason for the smaller association rate constant in the liposome-liposome system is the repulsive hydration effect. The effect of the surface density of the lectin immobilized on the liposome on the recognition was also examined.     

Mutual recognition between polymerized liposomes: enzyme and enzyme inhibitor system

In order to examine the usefulness of polymerized liposomes as a model for cell membranes, a mutual recognition phenomenon between different liposomes on which complementary ligands were attached was examined. We used trypsin- and soybean trypsin inhibitor (STI)-carrying polymerized liposomes to attain high sensitivities. The STI which was immobilized on the polymerized mono-dienoylphosphatidylcholine liposome showed a definite inhibitory effect on the catalytic activity of the trypsin which was immobilized on another polymerized liposome, whereas the inhibitory effect of the STI which was immobilized on the di-dienoylphosphatidylcholine liposome was much smaller than that of the mono-dienoylphosphatidylcholine system because of the larger rigity of the di-dienoylphosphatidylcholine liposome. These results suggest that the mutual recognition between complementary ligands can be realized by using polymerized liposomes with a physical stability and moderate deformability as their carriers.     

脂质体相行为对亲和素与膜表面模型受体特异性结合的影响

脂质体相行为对亲和素与膜表面模型受体特异性结合的影响刘铮,文辰晖,隋森芳（清华大学生物科学与技术系,北京１０００８４）关键词脂质体;抗生物素蛋白;荧光滴定生物体内的细胞是由一层磷脂双分子层的细胞膜包被的,细胞膜上有许多不同种类的受体,在细胞膜所执行的...     

Phosphoinositide-containing polymerized liposomes: stable membrane-mimetic vesicles for protein-lipid binding analysis

Stable phosphoinositide (PIP(n))-containing liposomes were prepared using polydiacetylene photochemistry. Tethered pentacosadiynyl inositol polyphosphate (InsP(n)) analogues of Ins(1,3,4)P(3), Ins(1,4,5)P(3), and Ins(1,3,4,5)P(4) were synthesized, incorporated into vesicles made up of diyne-phosphatidylcholine and -phosphatidylethanolamine, and polymerized by UV irradiation. The polymerized liposome nanoparticles showed markedly increased stability over conventional PIP(n)-containing vesicles as a result of the covalent conjugated ene-yne network in the acyl chains. The polymerized liposomes were specifically recognized by PIP(n) binding PH domains in liposome overlay assays and amplified luminescent proximity homogeneous assays. Moreover, the biotin moiety allowed attachment of the nanoparticles to a streptavidin-coated sensor chips in surface plasmon resonance (SPR) sensor. The PIP(n) headgroups displayed on SPR sensors showed higher affinities for PH domains and PIP(n) monoclonal antibodies than did monomeric PIP(n)-analogues with biotinylated acyl chains.     

Aggregation of ligand-modified liposomes by specific interactions with proteins. I: Biotinylated liposomes and avidin

The aggregation of biotin-modified phospholipid vesicles (liposomes) induced by binding the protein avidin in solution is analyzed experimentally and theoretically. Avidin has four binding sites that can recognize biotin specifically, and is able to cross-link the liposomes to form large aggregates. The aggregation kinetics were followed using quasi-elastic light scattering (QLS) to measure the mean particle size, and by measuring the solution turbidity. The rate and extent of aggregation were determined as a function of vesicle concentration, protein concentration, and the biotin density on the surface of the liposomes. A model based on Smoluchowski kinetics, fractal concepts, and Rayleigh and Mie light scattering theory was developed to analyze the experimental observations. Small aggregates (<7800 A diameter) may be treated as globular; however, the fractal nature of larger particles must be taken into account. Parameters in the model are taken from molecular simulations, or fit to the experimental observations. The aggregation kinetics are primarily determined by the biotin density on the liposome surface, the stoichiometric ratio of avidin molecules to liposomes, and the liposome concentration. Good agreement is found between the model and the experimental results. (c) 1996 John Wiley & Sons, Inc.     

Colorimetric competitive inhibition method for the quantification of avidin, streptavidin and biotin.

A colorimetric competitive inhibition assay for avidin, streptavidin and biotin was developed. The method for avidin or streptavidin was based on the competitive binding between avidin or streptavidin and a streptavidin-enzyme conjugate for biotinylated dextrin immobilized on the surface of a microtitre plate. For biotin quantitation the competition is between free biotin and the immobilized biotin for the streptavidin-enzyme conjugate. The limits of detection which was determined as the concentration of competitor required to give 90% of maximal absorbency (100% inhibition) was approximately 20 ng/100 microl per assay for avidin and streptavidin and 0.4 pg/100 microl per assay for biotin. The methods are simple, rapid, highly sensitive and adaptable to high throughput analysis.     

Generic liposome reagent for immunoassays

We have derivatized liposomes with antibodies by using avidin to crosslink biotinylated phospholipid molecules in the liposome membranes with biotinylated antibody molecules. A comparison of the biotin binding activity of avidin in solution and avidin associated with liposomes shows that avidin bound to biotinylated phospholipid in liposome membranes retains full binding activity for additional biotin molecules. Changes in the fluorescence spectrum of avidin have been used to characterize the binding capacity of avidin for biotin in solution, and change in intensity of light scattered due to aggregation of liposomes was used to measure the biotin binding activity of avidin associated with liposomes. Relative amounts of the biotinylated phospholipid, avidin, and biotinylated antibody have been optimized to produce stable liposomes which are derivatized with up to 1.7 nmol of antibody/mumol of lipid. These derivatized liposomes are highly reactive to immunospecific aggregation in the presence of multivalent antigen. A linear increase in light scattering was recorded between 1 and 10 pmol of antigen. This work shows that liposomes containing biotinylated phospholipid can be a successful generic reagent for immunoassays.     

A spin label study of egg white avidin.

Avidin is a tetrametric protein (mass 68,000 daltons) that binds 4 molecules of vitamin biotin (1). The biotin binding sites, 1 per subunit, are grouped in two pairs at opposite ends of the avidin molecule (GREEN, N.M., KONIECZNY, L., TOMS, E.J., and VALENTINE, R.C. (1971) Biochem. J. 125, 781). We have studied the topography of the avidin binding sites with the aid of four spin-labeled analogs of biotin: 4-biotinamido-2,2,6,6-tetramethyl-1-piperidinyloxy (II), 3-biotinamido-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (III), 3-biotinamidomethyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IV), 4-(biotinylglycyl)-amino-2,2,6,6-tetramethyl-1-piperidinyloxy (V). Fluorescence and optical absorption spectroscopy indicated that II to V occupied the same binding sites on avidin as did biotin. The electron spin resonance spectrum of the 4:1 complex between II and avidin contained broad line components characteristic of a highly immobilized spin label. Dipole-dipole interactions between spin labels bound to adjacent sites split each of the three major hyperfine lines into doublets with a separation of 13.8 G. The distance between adjacent bound nitroxide groups was calculated from this splitting to be 16 A. The dissociation of the 4:1 complex between II and avidin was biphasic with approximately half of the labels dissociating at a rate (kdiss equal to 2.51 times 10- minus 4 s- minus 1) that was much faster than the remainder (kdiss equal to 1.22 times 10- minus 5 s- minus 1). The electron spin resonance spectrum of the 2:1 complex between II and avidin clearly showed that, immediately after mixing, the spin labels were distributed in a random fashion among the available binding sites but that they slowly redistributed themselves so that each label bound to a site which was adjacent to an unoccupied site. The final time-independent electron spin resonance spectrum exhibited a splitting 69 G between the low and high field hyperfine lines which is characteristic of a highly immobilized, noninteracting spin label. Spin labels III and IV interacted with avidin in a similar fashion to that described for II with the exception that their dipolar splittings were 11.9 G and 14.2 G, respectively. From these splittings it was estimated that the distance between adjacent avidin-bound nitroxides was 16.7 A for labeled III and 15.7 A for label IV. The electron spin resonance spectrum of label V bound to avidin was characteristic of a noninteracting highly immobilized nitroxide with a maximum splitting of 62 G. The spectrum of V bound to avidin was independent of both time and the amount of bound label. The rate of dissociation of V from a 4:1 complex with avidin was monophasic. A model is proposed in which the recognition site for the heterocyclic ring system of biotin is represented as a cleft located within a hydrophobic depression in the surface of avidin.     

Avidin-biotin interactions at vesicle surfaces: adsorption and binding, cross-bridge formation, and lateral interactions.

Densely packed domains of membrane proteins are important structures in cellular processes that involve ligand-receptor binding, receptor-mediated adhesion, and macromolecule aggregation. We have used the biotin-avidin interaction at lipid vesicle surfaces to mimic these processes, including the influence of a surface grafted polymer, polyethyleneglycol (PEG). Single vesicles were manipulated by micropipette in solutions of fluorescently labeled avidin to measure the rate and give an estimate of the amount of avidin binding to a biotinylated vesicle as a function of surface biotin concentration and surface-grafted PEG as PEG-lipid. The rate of avidin adsorption was found to be four times less with 2 mol% PEG750 than for the unmodified surface, and 10 mol% PEG completely inhibited binding of avidin to biotin for a 2-min incubation. Using two micropipettes, an avidin-coated vesicle was presented to a biotinylated vesicle. In this vesicle-vesicle adhesion test, the accumulation of avidin in the contact zone was observed, again by using fluorescent avidin. More importantly, by controlling the vesicle membrane tension, this adhesion test provided a direct measure of the spreading pressure of the biotin-avidin-biotin cross-bridges confined in the contact zone. Assuming ideality, this spreading pressure gives the concentration of avidin cross-bridges in the contact zone. The rate of cross-bridge accumulation was consistent with the diffusion of the lipid-linked "receptors" into the contact zone. Once adherent, the membranes failed in tension before they could be peeled apart. PEG750 did not influence the mechanical equilibrium because it was not compressed in the contact zone, but it did perform an important function by eliminating all nonspecific adhesion. This vesicle-vesicle adhesion experiment, with a lower tension limit of 0.01 dyn/cm, now provides a new and useful method with which to measure the spreading pressures and therefore colligative properties of a range of membrane-bound macromolecules.     

Surface membrane biotinylation efficiently mediates the endocytosis of avidin bioconjugates into nucleated cells

Here we demonstrate that biotin covalently attached to cell surface obligates existing receptors to endocytose avidin bioconjugates into nucleated cells. Incubation of fluorescein-labeled avidin with biotinylated cell lines resulted in uniform and rapid surface attachment and endocytosis compared with no detectable association of the avidin-conjugated dye with unbiotinylated cells. Uptake was detected within minutes with efficiencies approaching 100% in cell lines and freshly obtained peripheral blood mononuclear cells. After 24 h, avidin was barely detectable on the surface of the nucleated cells. In marked contrast, fluorescent avidin remained exclusively on the external membrane of erythrocytes after 24 h. To investigate biotin-mediated endocytosis for the delivery of DNA, we prepared polyethylenimine-avidin (PEI-avidin) conjugates. Surface biotinylation significantly increased the transfection efficiencies of PEI-avidin condensed plasmid DNA coding green fluorescent protein (GFP) to the level of transferrin-receptor targeted gene delivery (15-20% GFP positive cells in culture after 48 h). The increase in transfection efficiency was blocked by the addition of free avidin or biotin to the culture medium. Biotin covalently bound to cell surface membrane proteins efficiently mediates the entry of avidin bioconjugates into nucleated cells.     

A radioimmunoassay for chicken avidin. Comparison with a [14C]biotin-binding method.

A double-antibody solid-phase radioimmunoassay for chicken avidin is reported. Avidin was labelled with 125I by the chloramine-T method. The bound and free avidin were separated with a second antibody bound to a solid matrix. In the logit-log scale the standard curve was linear from 1-2 to 100-200ng of avidin/ml. Cross-reaction of ovalbumin was less than 0.015%. Saturation of biotin-binding sites of avidin with an excess of biotin decreased radioimmunoassay values by about 15%. Recovery studies indicated that avidin can be assayed from all chicken tissues studied with radioimmunoassay, whereas the [14C]biotin/bentonite method gave poor recoveries for avidin in the liver and kidney. Radioimmunoassay and the [14C]biotin/bentonite method gave similar concentrations for oviduct avidin.     

Transport effects on the kinetics of protein-surface binding.

A detailed model is presented for protein binding to active surfaces, with application to the binding of avidin molecules to a biotin-functionalized fiber optic sensor in experiments reported by S. Zhao and W. M. Reichert (American Chemical Society Symposium Series 493, 1992). Kinetic data for binding in solution are used to assign an intrinsic catalytic rate coefficient k to the biotin-avidin pair, deconvoluted from transport and electrostatic factors via application of coagulation theory. This intrinsic chemical constant is built into a reaction-diffusion analysis of surface binding where activity is restricted to localized sites (representing immobilized biotin molecules). The analysis leads to an effective catalytic rate coefficient keff characterizing the active surface. Thereafter, solution of the transport problem describing absorption of avidin molecules by the macroscopic sensor surface leads to predictions of the avidin flux, which are found to be in good agreement with the experimental data. The analysis suggests the following conclusions. 1) Translational diffusion limitations are negligible for avidin-biotin binding in solution owing to the small (kinetically limiting) value k = 0.00045 m/s. 2) The sparse distribution of biotin molecules and the presence of a repulsive hydration force produce an effective surface-average catalytic rate coefficient keff of order 10(-7) m/s, much smaller than k. 3) Avidin binding to the fiber optic sensor occurs in an intermediate regime where the rate is influenced by both kinetics and diffusion.     

Fourier-transform infrared spectroscopic studies on avidin secondary structure and complexation with biotin and biotin-lipid assemblies.

Fourier-transform infrared studies have been carried out to investigate the secondary structure and thermal stability of hen egg white avidin and its complexes with biotin and with a biotinylated lipid derivative, N-biotinyl dimyristoyl phosphatidylethanolamine (DMBPE) in aqueous dispersion. Analysis of the amide I stretching band of avidin yielded a secondary structural content composed of approximately 66% beta-sheet and extended structures, with the remainder being attributed to disordered structure and beta-turns. Binding of biotin or specific association with the biotinylated lipid DMBPE did not result in any appreciable changes in the secondary structure content of the protein, but a change in hydrogen bond stability of the beta-sheet or extended chain regions was indicated. The latter effect was enhanced by surface interactions in the case of the biotin-lipid assemblies, as was demonstrated by electrostatic binding to a nonspecific negatively charged lipid. Difference spectra of the bound biotin implicated a direct involvement of the ureido moiety in the ligand interaction that was consistent with hydrogen bonding to amino acid residues in the avidin protein. It was found that complexation with avidin leads to a decrease in bond length of the biotin ureido carbonyl group that is consistent with a reduction of sp3 character of the C-O bond when it is hydrogen bonded to the protein. Studies of the temperature dependence of the spectra revealed that for avidin alone the secondary structure was unaltered up to approximately 75 degrees C, above which the protein undergoes a highly cooperative transition to an unfolded state with concomitant loss of ordered secondary structure. The complexes of avidin with both biotin and membrane-bound DMBPE lipid assemblies display a large increase in thermal stability compared with the native protein.     

Role of avidin and other biotin-binding proteins in the deposition and distribution of biotin in chicken eggs. Discovery of a new biotin-binding protein.

In addition to the previously characterized egg-yolk biotin-binding protein (BBP-I), we have discovered another BBP (BBP-II) in the plasma and yolk from laying hens. BBP-I is stable to 65 degrees C, whereas BBP-II is stable to 45 degrees C. Both proteins are normally saturated with biotin and together they account for most, if not all, of the biotin in hen plasma and yolk, except in hens fed excessive amounts of biotin (greater than 1 mg of biotin/kg of feed). The maximal production of BBP-I is attained at lower levels of dietary biotin (approximately 50 micrograms/kg) than for BBP-II (approximately 250 micrograms/kg); however, the maximal production of BBP-II is severalfold greater than for BBP-I. Consequently, as dietary biotin increases, the ratio of BBP-II to BBP-I increases and becomes constant at dietary intakes of biotin above 250 micrograms/kg. The observation that the amounts of these proteins are limited by biotin in the normal dietary range (less than 250 micrograms/kg) suggests that biotin is required for the synthesis, secretion or stability of these proteins. Although both plasma vitamin-protein complexes are transported to the oocyte and concentrated in the yolk, BBP-II is transferred more efficiently. Thus biotin deposition in the yolk is a function of the amounts and relative concentrations of the two proteins. Dietary biotin above 250 micrograms/kg exceeds the transport capacity of BBP-I and BBP-II in the plasma; however, unbound biotin does not accumulate. Rather it is efficiently scavenged by avidin in the oviduct and transferred to the egg albumen. Only when avidin becomes saturated at high dietary intake does free or weakly bound biotin accumulate in plasma and yolk. The synthesis of avidin is independent of dietary biotin. Small amounts of BBPs with the heat-stability of avidin or BBP-I respectively are present in the plasma of adult males or immature chickens. BBP-II, the major BBP in the plasma and yolk of laying hens, was not detected in the plasma of non-laying chickens.     

Ligand exchange between proteins. Exchange of biotin and biotin derivatives between avidin and streptavidin

We have studied the structural elements that affect ligand exchange between the two high affinity biotin-binding proteins, egg white avidin and its bacterial analogue, streptavidin. For this purpose, we have developed a simple assay based on the antipodal behavior of the two proteins toward hydrolysis of biotinyl p-nitrophenyl ester (BNP). The assay provided the experimental basis for these studies. It was found that biotin migrates unidirectionally from streptavidin to avidin. Conversely, the biotin derivative, BNP, is transferred in the opposite direction, from avidin to streptavidin. A previous crystallographic study (Huberman, T., Eisenberg-Domovich, Y., Gitlin, G., Kulik, T., Bayer, E. A., Wilchek, M., and Livnah, O. (2001) J. Biol. Chem. 276, 32031-32039) provided insight into a plausible explanation for these results. These data revealed that the non-hydrolyzable BNP analogue, biotinyl p-nitroanilide, was almost completely sheltered in streptavidin as opposed to avidin in which the disordered conformation of a critical loop resulted in the loss of several hydrogen bonds and concomitant exposure of the analogue to the solvent. In order to determine the minimal modification of the biotin molecule required to cause the disordered loop conformation, the structures of avidin and streptavidin were determined with norbiotin, homobiotin, and a common long-chain biotin derivative, biotinyl epsilon-aminocaproic acid. Six new crystal structures of the avidin and streptavidin complexes with the latter biotin analogues and derivatives were thus elucidated. It was found that extending the biotin side chain by a single CH(2) group (i.e. homobiotin) is sufficient to result in this remarkable conformational change in the loop of avidin. These results bear significant biotechnological importance, suggesting that complexes containing biotinylated probes with streptavidin would be more stable than those with avidin. These findings should be heeded when developing new drugs based on lead compounds because it is difficult to predict the structural and conformational consequences on the resultant protein-ligand interactions.     

Mutation of a critical tryptophan to lysine in avidin or streptavidin may explain why sea urchin fibropellin adopts an avidin-like domain

Sea urchin fibropellins are epidermal growth factor homologues that harbor a C-terminal domain, similar in sequence to hen egg-white avidin and bacterial streptavidin. The fibropellin sequence was used as a conceptual template for mutation of designated conserved tryptophan residues in the biotin-binding sites of the tetrameric proteins, avidin and streptavidin. Three different mutations of avidin, Trp-110-Lys, Trp-70-Arg and the double mutant, were expressed in a baculovirus-infected insect cell system. A mutant of streptavidin, Trp-120-Lys, was similarly expressed. The homologous tryptophan to lysine (W-->K) mutations of avidin and streptavidin were both capable of binding biotin and biotinylated material. Their affinity for the vitamin was, however, significantly reduced: from K(d) approximately 10(-15) M of the wild-type tetramer down to K(d) approximately 10(-8) M for both W-->K mutants. In fact, their binding to immobilized biotin matrices could be reversed by the presence of free biotin. The Trp-70-Arg mutant of avidin bound biotin very poorly and the double mutant (which emulates the fibropellin domain) failed to bind biotin at all. Using a gel filtration fast-protein liquid chromatography assay, both W-->K mutants were found to form stable dimers in solution. These findings may indicate that mimicry in the nature of the avidin sequence and fold by the fibropellins is not designed to generate biotin-binding, but may serve to secure an appropriate structure for facilitating dimerization.     

Aggregation of ligand-modified liposomes by specific interactions with proteins. II: Biotinylated liposomes and antibiotin antibody

The aggregation of biotinylated phospholipid vesicles (liposomes) cross-linked by antibiotin IgG was studied experimentally and theoretically. The liposomes were either low density liposomes that contained 0.4 mol% biotinylated phospholipid ( approximately 100 exposed biotin molecules per liposome), or high density liposomes that contained 2.7 mol% biotinylated phospholipid ( approximately 1000 exposed biotin molecules per liposome). The solution turbidity and mean particle size measured by quasi-elastic light scattering (QLS) were monitored throughout the aggregation. Three different lots of antibiotin antibodies, each with different association constants and binding heterogeneities, were used. The antibody binding characteristics affected the aggregation rates. The aggregation kinetics were analyzed using a model based on the Smoluchowski theory of aggregation, fractal concepts of aggregate microstructure, and Rayleigh and Mie light scattering theory. The experimental conditions of liposome concentration, protein concentration, and ligand density under which aggregation occurred correlated well with calculated sticking probabilities based on isotherms describing the adsorption of antibiotin antibody to the liposomes. These results are compared with prior observations made when avidin was used as the cross-linking protein. (c) 1996 John Wiley & Sons, Inc.     

Preparation and characterization of diacetylene polymerized liposomes for detection of autoantibodies

Affinity diacetylene liposomes were prepared with 10,12-tricosadiynoic acid and cardiolipin as the affinity ligand for anticardiolipin antibodies at a molar ratio of 80:20. Polymerization was carried out under UV irradiation, and the color transitions were monitored by visible absorption spectroscopy. Peaks at 635 nm (blue form), 540 nm (purple form), and 480 nm (red form) were observed as a function of time. These polymerized liposomes were used in a noncompetitive immunoassay for detection of anticardiolipin antibodies. Color changes were observed when reference serum containing specific immunoglobulin G, IgG, was added to polymerized liposome dispersions. The colorimetric signal due to IgG adsorption on the liposome surface was quantified as a colorimetric response defined as the change in percentage of blue color related to the initial percentage of blue color in the solutions. The colorimetric response was 10 times higher for specific IgG compared with nonspecific ones. These results suggest the unique potentialities of affinity diacetylene polymerized liposomes in the development of biosensors for diagnosis of autoimmune diseases.     

Disintegration of layer-by-layer assemblies composed of 2-iminobiotin-labeled poly(ethyleneimine) and avidin

A layer-by-layer thin film composed of avidin and 2-iminobiotin-labeled poly(ethyleneimine) (ib-PEI) was prepared and their sensitivity to the environmental pH and biotin was studied. The avidin/ib-PEI multilayer assemblies were stable at pH 8-12, whereas the assemblies were decomposed at pH 5-6 due to the low affinity of the protonated iminobiotin residue to avidin. The avidin/ib-PEI assemblies can be disintegrated upon addition of biotin and analogues in the solution as a result of the preferential binding of biotin or analogues to the binding site of avidin. The decomposition rate was arbitrarily controlled by changing the type of stimulant (biotin or analogues) and its concentration. The avidin/ib-PEI assemblies were disintegrated rapidly by the addition of biotin or desthiobiotin, whereas the rate of decomposition was rather slow upon addition of lipoic acid or 2-(4'-hydroxyphenylazo)benzoic acid. The present system may be useful for constructing the stimuli-sensitive devices that can release drug or other functional molecules.     

Preparation of ferritin-avidin conjugates by reductive alkylation for use in electron microscopic cytochemistry.

An improved technique was developed for the unidirectional covalent binding of avidin to ferritin by reductive alkylation. The method is based on the oxidation of sugar moieties on avidin and subsequent coupling to amino groups of ferritin via Schiff's bases followed by reduction with sodium borohydride. The resultant conjugate was used as an ultrastructural marker for the localization of surface receptor sites on biotin-derivatized whole cells. Erythrocytes were treated chemically with sodium meta-periodate and biotin hydrazide in succession. The ferritin-avidin conjugates were used to label the biotin sites either before or after fixation of the cells. The density and distribution of ferritin avidin conjugates on cell surfaces were anlyzed on thin sections and compared with those of cationized ferritin, which were shown to bind anionic sites of the erythrocyte membrane. The extensions of this method for the visualization of other systems is discussed.