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.     

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.     

Extremely high thermal stability of streptavidin and avidin upon biotin binding

The effect of biotin binding on the thermal stability of streptavidin (STV) and avidin (AVD) was evaluated using differential scanning calorimetry. Biotin binding increases the midpoint of temperature Tm of thermally induced denaturation of STV and AVD in phosphate buffer from 75 and 83 degrees C to 112 and 117 degrees C at full biotin saturation, respectively. This thermostability is the highest reported for proteins coming from either mesophilic or thermophilic organisms. In both proteins, biotin also increases the calorimetric enthalpy and the cooperativity of the unfolding. Thermal stability of STV was also evaluated in the presence of high concentrations of urea or guanidinium hydrochloride (GuHCl). In 6 M GuHCl, STV remains as a tetramer and the Tm of the STV-biotin complex is centered at 108 degrees C, a few degrees below the value obtained in phosphate buffer. On the contrary, STV under fully saturating condition remains mainly in its dimeric form in 8 M urea and the thermogram shows two endotherms. The main endotherm at a lower temperature has been ascribed to the dimeric liganded state with a Tm of 87 degrees C, and the higher temperature endotherm to the tetrameric liganded form with a Tm of 106 degrees C. As the thermostability of unliganded protein in the presence of urea is unchanged upon binding we related the extremely high thermal stability of this protein to both an increase in structural ordering and compactness with the preservation of the tetramer integrity.     

Evaluation of biotin-dye conjugates for use in an HPLC assay to assess relative binding of biotin derivatives with avidin and streptavidin

In this investigation, studies were conducted to determine if size exclusion HPLC could be used to assess relative association rates (on-rates) and dissociation rates (off-rates) of biotin derivatives from avidin (Av) and streptavidin (SAv). For easy detection and quantification of biotin derivatives, molecules that can be detected by UV absorbance were conjugated to biotin. Concern that conjugation of the chromophoric moieties (dyes) might affect biotin binding with Av and SAv or might interact with the HPLC column led to evaluation of 10 biotin-dye conjugates. The dyes conjugated with biotin included dansyl, cyanocobalamin (CN-Cbl), coumarin 343, Lissamine-rhodamine, fluorescein, Cascade Blue, Lucifer Yellow, Oregon Green, tetramethylrhodamine, and Alexa Fluor 594. The biotin-dye conjugates were initially evaluated to determine their peak characteristics on two different size exclusion HPLC columns. Measurement of the percent of biotin-dye conjugate bound with Av in the presence of an equal quantity of biotin provided an association rate relative to biotin. All of the biotin-dyes tested had association rates within a factor of 3x (slower) that of biotin. The relative dissociation rate of biotin-dye conjugates was assessed by challenging the biotin conjugate bound to Av or SAv with a large excess of biotin. All of the initial biotin-dye conjugates tested bound Av and SAv tightly resulting in very slow dissociation rates. From the biotin-dye conjugates studied, biotin-CN-Cbl, 6b, was selected as the best conjugate for the HPLC assay. To test the HPLC assay, an iminobiotin-CN-Cbl conjugate, 13a, and a biotin-sarcosine-CN-Cbl conjugate, 13b, were synthesized. The fact that the iminobiotin does not bind with Av at physiological pH was easily detected in the size exclusion HPLC assay. The biotin-sarcosine-CN-Cbl conjugate was expected to have a more rapid dissociation rate than the other biotin-dye conjugates. This was confirmed in that HPLC assay. Although 13b bound tightly with Av in the absence of added biotin, it was completely released within 1 h when challenged by an excess of biotin. A slower dissociation of 13b was noted with SAv. The results obtained indicate that CN-Cbl conjugates of biotin derivatives can be used to determine relative on-rates and off-rates of biotin derivatives with Av and SAv. The studies also demonstrated that the biotin-CN-Cbl conjugate, 6b, can be used as a reference compound to compare on-rates and off-rates of nonchromophoric biotin derivatives.     

Intermolecular interaction of avidin and PEGylated biotin

The equilibrium binding constants and stoichiometries between PEGylated biotins and avidin have been studied for a range of PEGylated biotin molecular weights. These studies show that as the molecular weight of PEG (polyethylene glycol) increases over the range 588, 3400, and 5000 g/mol, the equilibrium dissociation constants of PEGylated biotins with avidin increase to approximately 10 (-8) M compared with 10 (-15) M for the biotin-avidin complex. The stoichiometries of PEGylated biotins with avidin are 4:1 for 588 and 3400 g/mol PEG and 1:1 for 5000 g/mol PEG. The data demonstrate that the equilibrium binding constant and the stoichiometry of the avidin-biotin-PEG complex system can be adjusted by the length of PEG chains. This approach may be used with PEGylated biotin analogues for pretargeting in drug delivery, such as a biotin-PEGylated enzyme for converting an inactive prodrug into a cytotoxin. When a PEG chain is chosen as an appropriate spacer, the length of the PEG chain must be considered because PEG can block the binding sites on avidin.     

Biotin reagents for antibody pretargeting. 4. Selection of biotin conjugates for in vivo application based on their dissociation rate from avidin and streptavidin

An investigation was conducted to determine the affect of structural variation of biotin conjugates on their dissociation rates from Av and SAv. This information was sought to help identify optimal biotin derivatives for in vivo applications. Fifteen biotin derivatives were conjugated with a cyanocobalamin (CN-Cbl) derivative for evaluation of their "relative" dissociation rates by size exclusion HPLC analysis. Two biotin-CN-Cbl conjugates, one containing unaltered biotin and the other containing iminobiotin, were prepared as reference compounds for comparison purposes. The first structural variations studied involved modification of the biotinamide bond with a N-methyl moiety (i.e., sarcosine conjugate), lengthening the valeric acid side chain by a methylene unit (i.e., homobiotin), and replacing the biotinamide bond with thiourea bonds in two conjugates. The rate of dissociation of the biotin-CN-Cbl derivative from Av and SAv was significantly increased for biotin derivatives containing those structural features. Nine additional biotin conjugates were obtained by coupling amino acids or functional group protected amino acids to the biotin moiety. In the conjugates, the biotin moiety and biotinamide bond were not altered, but substituents of various sizes were introduced alpha to the biotinamide bond. The results obtained from HPLC analyses indicated that the rate of dissociation from Av or SAv was not affected by small substituents alpha to the biotinamide (e.g., methyl, hydroxymethyl, and carboxylate groups), but was significantly increased when larger functional groups were present. On the basis of the results obtained, it appears that biotin conjugates which retain an unmodified biotin moiety and have a linker molecule conjugated to it that has a small functional group (e.g., hydroxymethylene or carboxylate) alpha to the biotinamide bond are excellent candidates for in vivo applications. These structural features are obtained in the biotin amino acid conjugates: biotin-serine, biotin-aspartate, biotin-lysine, and biotin-cysteine. Importantly, these biotin derivatives can be readily conjugated with other molecules for specific in vivo applications. In our studies, these derivatives will be used in the design of new biotin conjugates to carry radionuclides for cancer therapy using the pretargeting approach.     

Optical rotatory dispersion, circular dichroism and far-ultraviolet spectra of avidin and streptavidin

1. The optical-rotatory-dispersion and circular-dichroism curves of avidin showed positive Cotton effects centred at 228mmu and 280mmu, close to the ultraviolet-absorption bands of tryptophan. These effects disappeared when avidin was dissociated into sub-units in guanidine hydrochloride. 2. Binding of biotin had only a small effect on the optical-rotatory-dispersion curve of avidin. 3. The absence of negative circular dichroism at wavelengths above 216mmu showed that there was little or no alpha-helix present in avidin. This interpretation was confirmed by Moffitt-Yang plots of the partial rotation due to the peptide bonds in the visible region of the spectrum. The calculated dispersion constants were remarkably similar to those of gamma-globulin and suggested the presence of peptide conformations other than alpha-helix and random coil. 4. The far-ultraviolet spectrum was also similar to that of gamma-globulin, the mean extinction coefficient of the peptide chromophore being much lower than the experimental value for a random-coil structure. 5. Streptavidin resembled avidin in showing two positive Cotton effects, but the negative dichroism below 220mmu suggested the presence of more alpha-helix than was found in avidin. Formation of the complex with biotin was accompanied by changes in rotation that were rather larger than those observed with avidin.     

Isolation of DNA-protein complexes based on streptavidin and biotin interaction

We describe a method for the purification of proteins binding to specific DNA sites based on the strong interaction between streptavidin and biotin. We tested the efficiency of this method using the Escherichia coli lactose operon operator-repressor system. dUTP coupled to biotin is incorporated into a DNA fragment containing the lactose operator. A crude E. coli extract is first incubated with the biotinylated fragment and the reaction mixture is filtered on a streptavidin-agarose column. Proteins retained on the column are either eluted alone by high salt or isopropyl beta-D-thiogalactoside, or as a complex with the DNA site by enzymatic digestion of the DNA. We thus obtained a 3400-fold enrichment of the repressor complexed to the operator in one step. The method is simple and makes use of commercially available reagents. The large concentration of biotin-binding sites of the streptavidin-agarose matrix (0.1 mumol/ml packed gel) provides a very high capacity for the concentration and purification of large amounts of proteins. The advantage of this method for the detection and purification of other DNA-binding proteins is discussed.     

Rapid estimation of avidin and streptavidin by fluorescence quenching or fluorescence polarization

A new biotin-carboxyfluorescein conjugate has been presented in the accompanying study (G. Kada et al., Biochim. Biophys. Acta 000 (1999) 000-000) which contains ethylene diamine as a 4-atom spacer. This so-called biotin-4-fluorescein showed exceptionally fast and tight binding to avidin and streptavidin, and binding was accompanied by strong quenching. In the present study the specific quenching of 'biotin-4-fluorescein' was utilized to measure (strept)avidin concentrations (0.2-2 nM) by the extent of fluorescence quenching at 8 nM ligand concentration. Adsorption of (strept)avidin to the assay tubes was suppressed by inclusion of bovine serum albumin (0.1 mg/ml). Virtually the same specific response to avidin and streptavidin was also observed with commercial 'fluorescein-biotin', except that >10 h incubation times were required. The slow association of 'fluorescein-biotin' was attributed to the anti-cooperative binding which is due to the much longer spacer as compared to 'biotin-4-fluorescein'. The third ligand tested in this study was 'biotin-4-FITC' which was analogous to 'biotin-4-fluorescein' except that carboxyfluorescein was replaced by the fluorescein isothiocyanate residue. Surprisingly, this probe was much less quenched by avidin but this was compensated by an exceptionally high fluorescence polarization in the avidin-bound state. In conclusion, the new ligand 'biotin-4-fluorescein' appeared to be the most general and convenient probe: quenching was most pronounced and linearly dependent on (strept)avidin concentrations, the dose response for streptavidin was almost the same as for avidin, and the association kinetics were fast enough to reach equilibrium within 30 min incubation time.     

Dimer-tetramer transition between solution and crystalline states of streptavidin and avidin mutants

The biotin-binding tetrameric proteins, streptavidin from Streptomyces avidinii and chicken egg white avidin, are excellent models for the study of subunit-subunit interactions of a multimeric protein. Efforts are thus being made to prepare mutated forms of streptavidin and avidin, which would form monomers or dimers, in order to examine their effect on quaternary structure and assembly. In the present communication, we compared the crystal structures of binding site W-->K mutations in streptavidin and avidin. In solution, both mutant proteins are known to form dimers, but upon crystallization, both formed tetramers with the same parameters as the native proteins. All of the intersubunit bonds were conserved, except for the hydrophobic interaction between biotin and the tryptophan that was replaced by lysine. In the crystal structure, the binding site of the mutated apo-avidin contains 3 molecules of structured water instead of the 5 contained in the native protein. The lysine side chain extends in a direction opposite that of the native tryptophan, the void being partially filled by an adjacent lysine residue. Nevertheless, the binding-site conformation observed for the mutant tetramer is an artificial consequence of crystal packing that would not be maintained in the solution-phase dimer. It appears that the dimer-tetramer transition may be concentration dependent, and the interaction among subunits obeys the law of mass action.     

A spectrophotometric assay for nanogram quantities of biotin and avidin

Parameters and conditions of an enzyme based assay for biotin and avidin are presented. Biotinylated glucose-6-phosphate dehydrogenase when complexed with avidin becomes inactivated. Thus it was possible to construct a competitive assay system for biotin. The assay is sensitive between 100-500 ng/ml and could detect as little as 10 ng in 0.1 ml with a between run error of 2.4%. It requires a 60 min incubation at 21 degrees C and 5 min to assay. The avidin assay, based on the degree of inactivation of biotinylated-glucose-6-phosphate dehydrogenase in relation to the concentration of avidin, could detect as little as 0.25 ng in 0.1 ml or 2.5 ng/ml with an assay time of 10 min with a between run error of 3.9%. Both assays are rapid with significant improvements over other non-isotopic methods in sensitivity and comparable to radioisotopic methods in sensitivity with the added advantage of ease of method.     

Cooperative biotin binding by streptavidin. Electrophoretic behavior and subunit association of streptavidin in the presence of 6 M urea

We describe the cooperativity in the biotin binding of streptavidin. We have developed an electrophoretic method which can separate streptavidin molecules with bound biotin from those without biotin. In 6 M urea, the electrophoretic mobility of streptavidin in polyacrylamide gels becomes significantly faster upon biotin binding. When streptavidin was titrated with biotin, only two major bands were observed on the gel, consisting of streptavidin molecules without bound biotin and those saturated with biotin. The change in mobility is due partly to the negative charge of the bound biotin, but it must reflect conformational changes of the protein molecule associated with biotin binding. Gel filtration chromatography showed that the streptavidin molecule dissociates into two subunit dimers in the presence of 6 M urea. These results suggest that the biotin binding by the streptavidin subunit dimer is cooperative and that some communication must exist between the two subunits.     

Expression of biotin-binding proteins,avidin and streptavidin,in plant tissues using plant vacuolar targeting sequences

Tobacco plants have been developed which constitutively express high levels of the biotin-binding proteins, avidin and streptavidin. These plants were phenotypically normal and produced fertile pollen and seeds. The transgene was expressed and its product located in the vacuoles of most cell types in the plants. Targeting was achieved by use of N-terminal vacuolar targeting sequences derived from potato proteinase inhibitors which are known to target constitutively to vacuoles in potato tubers and, under wound-induction, in tomato leaves. Avidin was located in protein body-like structures within the vacuole and transgene protein levels remained relatively constant throughout the lifetime of the leaf. We describe two chimeric constructs with similar levels of expression. One comprised a potato proteinase inhibitor I signal peptide cDNA sequence attached to an avidin cDNA and the second a potato proteinase inhibitor II signal peptide genomic sequence (including an intron) attached to a core streptavidin synthetic sequence. We were unable to regenerate plants when transformation used constructs lacking the targeting sequences. The highest levels observed (up to 1.5% of total leaf protein) confirm the vacuole as the organelle of choice for stable storage of plant-toxic transgene products. The efficient targeting of these proteins did not result in any measured changes in plant biotinmetabolism.     

Accurate measurement of avidin and streptavidin in crude biofluids with a new, optimized biotin-fluorescein conjugate

A new biotin-fluorescein conjugate with an ethylene diamine spacer was found to be the first fluorescent biotin derivative which truly mimicked d-biotin in terms of high affinity, fast association, and non-cooperative binding to avidin and streptavidin tetramers. These exceptional properties were attributed to the small size/length of the new ligand since all larger/longer biotin derivatives are known for their mutual steric hindrance and anti-cooperative binding in 4:1 complexes with avidin and streptavidin tetramers. Specific binding of the new biotin-fluorescein conjugate towards avidin and streptavidin was accompanied by 84-88% quenching of ligand fluorescence. In the accompanying study this effect was used for rapid estimation of avidin and streptavidin in a new 'single tube assay'. In the present study the strong quenching effect was utilized to accurately monitor stoichiometric titration of biotin-binding sites in samples with >/=200 pM avidin or streptavidin. The concentration was calculated from the consumption of fluorescent ligand up to the distinct breakpoint in the fluorescence titration profile which was marked by the abrupt appearance of strongly fluorescent ligands which were in excess. Due to this protocol the assay was not perturbed by background fluorescence or coloration in the unknown samples. The new fluorescence titration assay is particularly suited for quick checks on short notice because getting started only means to thaw an aliquot of a standardized stock solution of fluorescent ligand. No calibration is required for the individual assay and the ligand stock solution needs to be restandardized once per week (or once per year) when stored at -25 degrees C (or at -70 degrees C, respectively).     

Easily reversible desthiobiotin binding to streptavidin,avidin, and other biotin-binding proteins: uses for protein labeling,detection, and isolation

The high-affinity binding of biotin to avidin, streptavidin, and related proteins has been exploited for decades. However, a disadvantage of the biotin/biotin-binding protein interaction is that it is essentially irreversible under physiological conditions. Desthiobiotin is a biotin analogue that binds less tightly to biotin-binding proteins and is easily displaced by biotin. We synthesized an amine-reactive desthiobiotin derivative for labeling proteins and a desthiobiotin-agarose affinity matrix. Conjugates labeled with desthiobiotin are equivalent to their biotinylated counterparts in cell-staining and antigen-labeling applications. They also bind to streptavidin and other biotin-binding protein-based affinity columns and are recognized by anti-biotin antibodies. Fluorescent streptavidin conjugates saturated with desthiobiotin, but not biotin, bind to a cell-bound biotinylated target without further processing. Streptavidin-based ligands can be gently stripped from desthiobiotin-labeled targets with buffered biotin solutions. Thus, repeated probing with fluorescent streptavidin conjugates followed by enzyme-based detection is possible. In all applications, the desthiobiotin/biotin-binding protein complex is easily dissociated under physiological conditions by either biotin or desthiobiotin. Thus, our desthiobiotin-based reagents and techniques provide some distinct advantages over traditional 2-iminobiotin, monomeric avidin, or other affinity-based techniques.     

Bioluminescent assays using glucose-6-phosphate dehydrogenase: application to biotin and streptavidin detection

A streptavidin-glucose-6-phosphate dehydrogenase (G6PDH) conjugate was synthesized and its properties were studied, along with those of biotin-G6PDH conjugates. Two bioluminescent assays were used. Streptavidin was assayed in two steps: streptavidin samples were first incubated with a small amount of biotin-G6PDH and then with biotinylated rabbit gamma-globulins. The complex was immobilized on a bioluminescent immunoadsorbent. In the single-step biotin assay, free biotin was allowed to compete with biotin linked to rabbit gamma-globulins for binding to streptavidin-G6PDH in the presence of the bioluminescent immunoadsorbent. Neither assay required washing or separation steps and the sensitivity was 0.2 ng for streptavidin and 100 fg for biotin. Different applications are described: studies of biotin reactivity when linked to probes in solution or immobilized, and quantitation of biotin in biotinylated DNA probes and oligonucleotides.     

Chemical linkage to injected tissues is a distinctive property of oxidized avidin

We recently reported that the oxidized avidin, named AvidinOX®, resides for weeks within injected tissues as a consequence of the formation of Schiff''s bases between its aldehyde groups and tissue protein amino groups. We also showed, in a mouse pre-clinical model, the usefulness of AvidinOX for the delivery of radiolabeled biotin to inoperable tumors. Taking into account that AvidinOX is the first oxidized glycoprotein known to chemically link to injected tissues, we tested in the mouse a panel of additional oxidized glycoproteins, with the aim of investigating the phenomenon. We produced oxidized ovalbumin and mannosylated streptavidin which share with avidin glycosylation pattern and tetrameric structure, respectively and found that neither of them linked significantly to cells in vitro nor to injected tissues in vivo, despite the presence of functional aldehyde groups. The study, extended to additional oxidized glycoproteins, showed that the in vivo chemical conjugation is a distinctive property of the oxidized avidin. Relevance of the high cationic charge of avidin into the stable linkage of AvidinOX to tissues is demonstrated as the oxidized acetylated avidin lost the property. Plasmon resonance on matrix proteins and cellular impedance analyses showed in vitro that avidin exhibits a peculiar interaction with proteins and cells that allows the formation of highly stable Schiff''s bases, after oxidation.