Site-specific photobiotinylation of antibodies, light chains, and immunoglobulin fragments

The high affinity of biotin for avidin has been exploited for many antibody-based assays. This requires that biotin is covalently conjugated to the antibody molecule. Several chemically reactive biotinylation reagents are commercially available. Except for the attachment via sulfhydryl groups in the immunoglobulin (Ig) molecule, these reagents attach biotin randomly to various amino acid side chains. Although non-site-specific modification of antibodies does not interfere in most immunoassays, specific application and sensitive antibodies would benefit from site-specific biotinylation. Here we describe an affinity biotinylation technique based on a photoreactive biotin reagent. The design of this reaction was possible from the discovery of a conserved binding site in the variable Ig domain for nucleotides and nucleosides. The described photoaffinity biotinylation offers the advantages of ease, convenience, and production of a reproducible and defined biotinylated antibody preparation.     

Enhancing the thermal stability of avidin. Introduction of disulfide bridges between subunit interfaces

In this study we showed that tetrameric chicken avidin can be stabilized by introducing intermonomeric disulfide bridges between its subunits. These covalent bonds had no major effects on the biotin binding properties of the respective mutants. Moreover, one of the mutants (Avd-ccci) maintained its tetrameric integrity even in denaturing conditions. The new avidin forms Avd-ci and Avd-ccci, which have native --> denatured transition midpoints (T(m)) of 98.6 and 94.7 degrees C, respectively, in the absence of biotin, will find use in applications where extreme stability or minimal leakage of subunits is required. Furthermore, we showed that the intramonomeric disulfide bridges found in the wild-type avidin affect its stability. The mutant Avd-nc, in which this bridge was removed, had a lower T(m) in the absence of biotin than the wild-type avidin but showed comparable stability in the presence of biotin.     

Purification and crystallization of avidin

An improved purification, including crystallization, of avidin from hen's-egg white is described. The product bound 15.1mug of biotin/mg, corresponding to an equivalent weight of 16200. The amino acid composition showed an integral number of amino acid residues per 16200g, which suggested that each molecule of avidin (mol. wt. approx. 66000) contained four identical subunits.     

The effect of N-bromosuccinimide on the sub-unit structure of avidin and its complexes with biotin

1. Each molecule of biotin bound to avidin protected four tryptophan residues from oxidation by N-bromosuccinimide, regardless of the occupancy of neighbouring binding sites in the four-sub-unit avidin molecule. 2. The oxidation products from avidin molecules in which some of the sites were occupied were separated on columns of Sephadex G-100. In the absence of biotin, oxidized avidin broke down into sub-units, which partly aggregated. When some of the sites were occupied by biotin, the only detectable products were completely oxidized avidin (sub-units and large aggregates) and unoxidized avidin-biotin complex (tetramer). Since the biotin-containing sub-units were randomly distributed before oxidation took place, they must have dissociated from the molecules containing oxidized sub-units and then reassociated to form the tetrameric avidin-biotin complex. 3. This reassociation still occurred in 3.5m-guanidinium chloride, which prevents the reassociation of unoccupied sub-units. During their brief existence in this medium, the sub-units of avidin-biotin complex were protected from oxidation by N-bromo-succinimide to the same extent as was the tetrameric complex. 4. It is concluded that sub-units of avidin-biotin complex do not readily lose their biotin, even in 3.5m-guanidinium chloride, and that monomeric biotin-binding species are probably present in solutions of avidin sub-units at guanidinium chloride concentrations between 3.0m and 3.5m.     

Fluorogen-Activating scFv Biosensors Target Surface Markers on Live Cells Via Streptavidin or Single-Chain Avidin

Fluorescence biosensors are indispensable tools for understanding protein behavior and function in cells. Recent advancements utilize fluorogen-activating-proteins (FAPs) that form complexes with small organic molecules (fluorogens) and result in their fluorescence activation. The technology has found multiple uses in protein discovery applications; however, the current method of detection requires the expression of FAPs as gene fusion tags in cells—a process that is time- and labor-intensive. In this report, we present an alternate method that utilizes FAPs as affinity reagents. Accordingly, we isolated soluble reagents based on FAP fusions with streptavidin (Strep) or avidin proteins, both highly selective for biotin. When tested in vitro, the reagents displayed bi-functional activity, fluorogen activation, and biotin affinity. For live-cell protein discovery, surface targets were biotinylated via biotin-tagged immunoglobulins or a genetically encoded biotin acceptor peptide. As a result, when the cells were labeled with FAP–Strep or FAP–avidin reagent, the in vivo fluorescence measurements indicated high target specificity, minimal background, and bright signal detection. In summary, we present a novel FAP reagent platform that offers a rapid and efficient approach for cell surface protein detection.     

Identification of avidin and streptavidin binding motifs among peptides selected from a synthetic peptide library consisting solely of D-amino acids

Peptides consisting solely of D -amino acids (D -peptides) as opposed to their L -counterparts (L -peptides) are resistant towards proteolytic degradation in the organism and may therefore be useful in future efforts to develop new stable peptide-based drugs. Using the random synthetic peptide library technique several L - and D -peptides, capable of binding to both avidin and streptavidin, were found. The L -peptides contained the previously described HPQ/M motifis, and among the D -peptides three binding motifs could be identified, of which the most frequently found one contained an N-terminal aliphatic hydrophobic amino acid (V, L or I) and an aromatic amino acid (Y or F) on the second position. At the third position in this motif several different amino acid residues were found, although N was the most frequent. Peptides representing two of the D -motifs were synthesized as well as peptides containing the HPQ/M motifs, and their binding properties were examined. Although the D -peptides were originally selected using avidin they also inhibited binding between immobilized biotin and soluble streptavidin as well as avidin. The IC₅₀ of some of the peptides were approximately 10⁵ times higher than the IC₅₀ for biotin but some had a lower IC₅₀ than iminobiotin. The D -peptides, which were originally selected from the library using avidin, could also inhibit the binding between streptavidin and biotin. Likewise, L -peptides selected from a library screened with streptavidin, could inhibit the binding of both streptavidin and avidin to immobilized biotin. Furthermore, the D -peptide, VFSVQSGS, as well as biotin could inhibit binding of streptavidin to an immobilized L -peptide (RYHPQSGS). This indicates that the biotin-like structure mimicked by these two seemingly very different peptides may react with the same binding sites in the streptavidin molecule.     

Solid-phase biotinylation of antibodies

Biotinylation is an established method of labeling antibody molecules for several applications in life science research. Antibody functional groups such as amines, cis hydroxyls in carbohydrates or sulfhydryls may be modified with a variety of biotinylation reagents. Solution-based biotinylation is accomplished by incubating antibody in an appropriate buffered solution with biotinylation reagent. Unreacted biotinylation reagent must be removed via dialysis, diafiltration or desalting. Disadvantages of the solution-based approach include dilution and loss of antibody during post-reaction purification steps, and difficulty in biotinylation and recovery of small amounts of antibody. Solid-phase antibody biotinylation exploits the affinity of mammalian IgG-class antibodies for nickel IMAC (immobilized metal affinity chromatography) supports. In this method, antibody is immobilized on a nickel-chelated chromatography support and derivitized on-column. Excess reagents are easily washed away following reaction, and biotinylated IgG molecule is recovered under mild elution conditions. Successful solid phase labeling of antibodies through both amine and sulfhydryl groups is reported, in both column and mini-spin column formats. Human or goat IgG was bound to a Ni-IDA support. For sulfhydryl labeling, native disulfide bonds were reduced with TCEP, and reduced IgG was biotinylated with maleimide-PEO(2) biotin. For amine labeling, immobilized human IgG was incubated with a solution of NHS-PEO(4) biotin. Biotinylated IgG was eluted from the columns using a buffered 0.2 M imidazole solution and characterized by ELISA, HABA/avidin assay, probing with a streptavidin-alkaline phosphatase conjugate, and binding to a monomeric avidin column. The solid phase protocol for sulfhydryl labeling is significantly shorter than the corresponding solution phase method. Biotinylation in solid phase is convenient, efficient and easily applicable to small amounts of antibody (e.g. 100 microg). Antibody biotinylated on-column was found to be equivalent in stability and antigen-recognition ability to antibody biotinylated in solution. Solid-phase methods utilizing Ni-IDA resin have potential for labeling nucleic acids, histidine-rich proteins and recombinant proteins containing polyhistidine purification tags, and may also be applicable for other affinity systems and labels.     

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.     

Biotin-conjugated reagents as site-specific probes of membrane protein structure: application to the study of the human erythrocyte hexose transporter

A novel labeling procedure using biotin-conjugated protein-modifying reagents has been employed to study the structure and function of the human erythrocyte hexose transporter. The carbohydrate moiety of the isolated, reconstituted transporter was labeled by using galactose oxidase/biotin hydrazide. Cysteine residues, which are essential for transporter function, were tagged with a biotin-conjugated maleimide. Labeling with this reagent inhibited the binding of cytochalasin B to the transporter. Following sodium dodecyl sulfate-gel electrophoresis, labeling of the transporter and its proteolytic fragments was detected by Western blotting and probing with alkaline phosphatase-conjugated avidin. After tryptic cleavage of the transporter into two membrane domains, preparations reacted with galactose oxidase/biotin hydrazide were labeled on the 25-kDa glycosylated fragment, but not on the carbohydrate-free 19-kDa peptide. Biotin-maleimide-labeled cysteine residues on both peptides. Transporter polypeptide was fragmented more extensively using Staphylococcus aureus V8 protease. Limited digestion produced a broad band of 30-50 kDa and sharper bands of 23 and 21 kDa. More extensive digestion resulted in the disappearance of the 23-kDa peptide and the appearance of sharp bands of 20, 19, 17, 13, 11, 8, and 7 kDa. Biotin label introduced with galactose oxidase/biotin hydrazide was found on the broad 30-kDa band, confirming its identity as a glycopeptide. All of the peptides weighing more than 11 kDa contained cysteine residues labeled with biotin maleimide, while the 8- and 7-kDa peptides were unlabeled. These results demonstrate the potential usefulness of biotin-conjugated reagents as site-specific probes of membrane protein structure.     

Direct effects of radiation on the avidin-biotin system. Absence of energy transfer

Frozen solutions of biotinylated glucose-6-phosphate dehydrogenase and fluorescently tagged avidin were exposed to high energy ionizing radiation. Parallel experiments with peroxidase coupled to streptavidin and with biotinylated phycoerythrin were also performed. The loss of function of each compound was analyzed according to target theory. Target analysis revealed that the radiation-sensitive mass associated with the enzymatic activity and that associated with the fluorescence were unchanged by irradiation in the strongly coupled state. Therefore the noncovalent bonds between biotin and avidin do not permit the transfer of radiation-deposited energy in amounts sufficient to destroy the activity of apposing molecule.     

Biotinylphallotoxins: preparation and use as actin probes

We describe the synthesis of four phalloidin derivatives conjugated with biotin. An aminomethyldithiolane derivative of ketophalloidin was used as a reactive starter compound, and biotin residues were coupled to this molecule either directly, separated by spacer chains comprised of one or two glycyl residues, or of a 12-atom long chain constructed from succinic acid and hexamethylendiamine. Although all products still displayed a high affinity for F-actin, as seen in competition experiments with [3H]-demethylphalloidin, only the one with the longest spacer (BHPP) showed specific and high-affinity decoration of actin filaments in permeabilized cells, in conjunction with FITC-coupled avidin and fluorescence microscopy. Combined with gold-streptavidin, BHPP decorated the actin filament system at the light and electron microscopic level faithfully and with satisfactory density. Actin filaments polymerized in vitro from purified protein were not as densely labeled as had been expected. However, in all these experiments the new phalloidin probe, when combined with avidin or streptavidin, yielded clear and highly specific labeling of F-actin. Therefore, this system is useful to identify and localize actin unambiguously in microfilaments, independent of actin antibodies, and should facilitate double-label experiments on cytoskeletal components at the ultrastructural level.     

Affinity chromatography of DNA labeled with chemically cleavable biotinylated nucleotide analogs

DNA labeled with the chemically cleavable biotinylated nucleotide Bio-12-SS-dUTP was chromatographed on biotin cellulose affinity columns using either avidin or streptavidin as the affinity reagent. Although both proteins were equally effective in binding the Bio-12-SS-DNA to the affinity resin, two important differences were found. First, nonbiotinylated DNA bound to avidin, but not to streptavidin, in buffers containing 50 mM NaCl. Second, Bio-12-SS-DNA was released much more slowly from the streptavidin affinity column than from the avidin column upon washing with buffer containing dithiothreitol. This difficulty in reducing the disulfide bond of Bio-12-SS-DNA bound to streptavidin is most likely due to steric protection of the disulfide bond by the protein. This conclusion is supported by our finding that DNA labeled with another biotinylated nucleotide analog, Bio-19-SS-dUTP, is rapidly and efficiently recovered from a streptavidin column. In Bio-19-SS-DNA, the distance between the disulfide bond and the biotin group is approximately 10 A greater than that in Bio-12-SS-DNA. Therefore, Bio-19-SS-dUTP and streptavidin form the basis of an efficient affinity system for the isolation and subsequent recovery of biotinylated DNA in the presence of low ionic strength buffers.     

DNA condensation by high-affinity interaction with avidin

Avidin, the basic biotin-binding glycoprotein from chicken egg white, is known to interact with DNA, whereas streptavidin, its neutral non-glycosylated bacterial analog, does not. In the present study we investigated the DNA-binding properties of avidin. Its affinity for DNA in the presence and absence of biotin was compared with that of other positively charged molecules, namely the protein lysozyme, the cationic polymers polylysine and polyarginine and an avidin derivative with higher isoelectric point (pI approximately 11) in which most of the lysine residues were converted to homoarginines. Gel-shift assays, transmission electron microscopy and dynamic light scattering experiments demonstrated an unexpectedly strong interaction between avidin and DNA. The most pronounced gel-shift retardation occurred with the avidin-biotin complex, followed by avidin alone and then guanidylated avidin. Furthermore, ultrastructural and light-scattering studies showed that avidin assembles on the DNA molecule in an organized manner. The assembly leads to the formation of nanoparticles that are about 50-100 nm in size (DNA approximately 5 kb) and have a rod-like or toroidal shape. In these particles the DNA is highly condensed and one avidin is bound to each 18 +/- 4 DNA base pairs. The complexes are very stable even at high dilution ([DNA] =10 pM) and are not disrupted in the presence of buffers or salt (up to 200 mM NaCl). The other positively charged molecules also condense DNA and form particles with a globular shape. However, in this case, these particles disassemble by dilution or in the presence of low salt concentration. The results indicate that the interaction of avidin with DNA may also occur under physiological conditions, further enhanced by the presence of biotin. This DNA-binding property of avidin may thus shed light on a potentially new physiological role for the protein in its natural environment.     

Neoglycoproteins: preparation and properties of complexes of biotinylated asparagine-oligosaccharides with avidin and streptavidin

Neoglycoproteins in which the oligosaccharide moieties are attached noncovalently to the protein through a high-affinity ligand have been prepared from biotinylated oligosaccharides and avidin or the nonglycosylated microbial analogue streptavidin. One of the asparagine-oligosaccharides purified from Pronase-digested ovalbumin (Man6-GlcNAc2-Asn) was reacted with an excess of the hydroxysuccinimide ester of biotin or, for the purpose of quantitation, [3H]biotin. Derivatives were also prepared with an extension "arm", a 6-aminohexanoyl group, between biotin and asparagine. When the purified biotinyl-Asn-oligosaccharide was added to avidin or streptavidin, a complex was formed containing 3 mol of oligosaccharide/mol of protein. The complexes were stable at neutral pH in the absence of biotin and could be dialyzed for 2 weeks without any significant loss of ligand. In the presence of biotin, or under denaturing conditions, the oligosaccharide derivative was released and could be quantitatively recovered. To assess the influence of the protein matrix on the reactivity of the oligosaccharide units, free biotinyl-Asn-oligosaccharide and the corresponding avidin and streptavidin complexes were exposed to alpha-mannosidase in parallel experiments. The rate of hydrolysis of the free derivative was severalfold faster than that of the two protein complexes, and at the time when about 90% of the free derivative had all five alpha-mannosyl residues removed, the majority of the protein-bound derivatives contained two to four undigested alpha-mannosyl residues and also had a significant amount of undigested starting material. The ease of preparation and the properties of these neoglycoproteins suggest that they should be excellent models for the study of glycoprotein-receptor binding and glycoprotein processing.     

Biotin derivatives of D-Phe-Pro-Arg-CH2Cl for active-site-specific labeling of thrombin and other serine proteinases

Biotin derivatives of peptide chloromethyl ketones have ideal properties for specific labeling of the catalytic sites of serine proteinases but have not been widely used as probes because of the difficulty of synthesis and their instability. To make the reagents more accessible, a simple, economical method was developed for preparation of three biotin derivatives of the thrombin-specific inhibitor D-Phe-Pro-Arg-CH2Cl containing increasing lengths of the spacer connecting biotin. Reaction of the peptide with biotin-succinimidyl esters and purification by conventional chromatography yielded the compounds in 91-96% purity. The biotin-labeled inhibitors bound avidin with stoichiometries of 0.88-1.02 mol biotin compound/mol avidin subunits and irreversibly inactivated human thrombin with stoichiometries of 0.89-1.10 mol inhibitor/mol thrombin. Comparison of the three inhibitors by Western blotting indicated that a > or = 7- to 14-atom spacer was needed for sensitive (approximately 10 ng) detection of thrombin, with the derivative lacking a spacer only weakly detected because of its greatly reduced affinity for avidin. Application of the compounds to identify catalytically active products of factor Xa-catalyzed human prethrombin 1 activation in the absence of the protein cofactor, factor Va, allowed the direct observation of transient, low levels of the active intermediate, meizothrombin des-fragment 1, in addition to thrombin. Formation of this intermediate is concluded to reflect an intrinsic property of factor Xa activation of prethrombin 1 that is modulated by factor Va. The methods developed for preparation and characterization of the biotin-labeled inhibitors may be applicable to other tripeptide chloromethyl ketones, and the reagents can be employed for labeling of serine proteinases of diverse substrate specificity.     

Biotin induces tetramerization of a recombinant monomeric avidin. A model for protein-protein interactions

Chicken avidin, a homotetramer that binds four molecules of biotin was converted to a monomeric form by successive mutations of interface residues to alanine. The major contribution to monomer formation was the mutation of two aspartic acid residues, which together account for ten hydrogen bonding interactions at the 1-4 interface. Mutation of these residues, together with the three hydrophobic residues at the 1-3 interface, led to stable monomer formation in the absence of biotin. Upon addition of biotin, the monomeric avidin reassociated to the tetramer, which exhibited properties similar to those of native avidin, with respect to biotin binding, thermostability, and protease resistance. To our knowledge, these unexpected results represent the first example of a small monovalent ligand that induces oligomerization of a monomeric protein. This study may suggest a biological role for low molecular weight ligands in inducing oligomerization and in maintaining the stability of multimeric protein assemblies.     

The properties of subunits of avidin coupled to Sepharose

Avidin that had been coupled to Sepharose 4B activated with CNBr retained over 90% of its biotin-binding capacity. When low concentrations of CNBr were used about 75% of the protein could be removed from the Sepharose by washing with guanidinium chloride (6 m). The remaining 25%, the covalently bound subunits, had an almost undiminished capacity for biotin but a decreased affinity. Addition of avidin subunits in guanidinium chloride to the coupled subunits followed by dilution or dialysis restored the original biotin-binding capacity and affinity. Three classes of binding sites were present in preparations of the subunits. About 25% were weak (K=5x10(-8)m), about one third exchanged their biotin in a few minutes (K approximately 10(-10)m) and the remainder were indistinguishable from the native tetramer. The last-named exchanged their bound biotin at a similar rate at pH5 and at pH2, they did not lose their biotin in 6 m-guanidinium chloride and they were resistant to tryptic digestion in the absence of biotin. The proportion of these stable sites could be increased to 65% when the subunits coupled to Sepharose were incubated at 37 degrees C. This increase was reversed by guanidinium chloride, which suggested that it was caused by a temperature-dependent association of covalently linked subunits. This in turn implies a temperature-dependent mobility of the agarose matrix of the Sepharose. Analysis of the spatial distribution of subunits within the Sepharose beads led to the conclusion that the association of subunits implied that they could move through distances greater than 20nm (several hundred A). This mobility and consequent formation of tetramer was greatly decreased when avidin subunits were coupled to Sepharose that had been cross-linked with divinyl sulphone.     

Biotin binding to avidin. Oligosaccharide side chain not required for ligand association.

A commercially available, purified preparation of avidin was found to comprise two polypeptide bands (Mr 18,000 and Mr 15,500 respectively). Both bands bound biotin as assessed by biotin overlays of protein blots. The Mr 15,500 polypeptide was found to differ from the Mr 18,000 polypeptide only in its sugar content. When the commercial preparation was applied to a concanavalin A affinity column, the glycosylated forms were retarded as expected, and homotypic nonglycosylated avidin tetramers which failed to bind selectively to the column were collected in the effluent. The biotin-binding properties of the nonglycosylated avidin were equivalent to those obtained for the native (glycosylated) avidin molecule, indicating that the oligosaccharide moiety is not essential for the binding activity.     

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.     

Crystal structure of soybean proglycinin A1aB1b homotrimer

Soybean glycinin is a member of the 11 S globulin family. The crystal structure of proglycinin was determined by X-ray crystallography at 2.8 A resolution with an R-factor of 0.199 and a free R-factor of 0.250. A trimer molecule was found in an asymmetric unit of crystals. The trimer model contains three A1aB1b subunits and comprises 1128 amino acid residues and 34 water molecules. The constituent protomers of the homo-trimeric protein are arranged around a 3-fold symmetry axis with dimensions of 95 Ax95 Ax40 A. The protomer model is composed of five fragments which correspond roughly to conserved regions based on the sequence alignment of various 11 S globulins. The core of the protomer consists of two jelly-roll beta-barrels and two extended helix domains. This structure of proglycinin is similar to those of canavalin and phaseolin belonging to the 7 S globulin family, strongly supporting the hypothesis that both 7 S and 11 S globulins are derived from a common ancestor. The inter and intra-chain disulfide bonds conserved in the 11 S globulin family are clearly observed. It is found that the face with the inter-chain disulfide bond (IE face) contains more hydrophobic residues than that with the intra-chain disulfide bond. This suggests that a mature hexamer is formed by the interaction between the IE faces after processing.