Dynamic force measurements of avidin-biotin and streptavdin-biotin interactions using AFM

Using atomic force microscopy (AFM) we performed dynamic force measurements of the adhesive forces in two model systems: avidin-biotin and streptavidin-biotin. In our experiments we used glutaraldehyde for immobilization of (strept)avidin on the tip and biotin on the sample surface. Such interface layers are more rigid than those usually reported in the literature for AFM studies, when (strept)avidin is coupled with biotinylated bovine albumin and biotin with agarose polymers. We determined the dependence of the rupture forces of avidin-biotin and streptavidin-biotin bonds in the range 300-9600 pN/s. The slope of a semilogarithmic plot of this relation changes at about 1700 pN/s. The existence of two different regimes indicates the presence of two activation barriers of these complexes during the dissociation process. The dissociation rates and activation energy barriers, calculated from the Bell model, for the avidin-biotin and streptavidin-biotin interactions are similar to each other for loading rates > 1700 pN/s but they are different from each other for loading rates < 1700 pN/s. In the latter case, the dissociation rates show a higher stability of the avidin-biotin complex than the streptavidin-biotin complex due to a larger outer activation barrier of 0.8 k(B)T. The bond-rupture force is about 20 pN higher for the avidin-biotin pair than for the streptavidin-biotin pair for loading rates < 1700 pN/s. These two experimental observations are in agreement with the known structural differences between the biotin binding pocket of avidin and of streptavidin.     

Biochemical and Biological Characterization of a New Oxidized Avidin with Enhanced Tissue Binding Properties

Chicken avidin and bacterial streptavidin are widely employed in vitro for their capacity to bind biotin, but their pharmacokinetics and immunological properties are not always optimal, thereby limiting their use in medical treatments. Here we investigate the biochemical and biological properties of a new modified avidin, obtained by ligand-assisted sodium periodate oxidation of avidin. This method allows protection of biotin-binding sites of avidin from inactivation caused by the oxidation step and delay of avidin clearance from injected tissue by generation of aldehyde groups from avidin carbohydrate moieties. Oxidized avidin shows spectroscopic properties similar to that of native avidin, indicating that tryptophan residues are spared from oxidation damage. In strict agreement with these results, circular dichroism and isothermal titration calorimetry analyses confirm that the ligand-assisted oxidation preserves the avidin protein structure and its biotin binding capacity. In vitro cell binding and in vivo tissue residence experiments demonstrate that aldehyde groups provide oxidized avidin the property to bind cellular and interstitial protein amino groups through Schiff''s base formation, resulting in a tissue half-life of 2 weeks, compared with 2 h of native avidin. In addition, the efficient uptake of the intravenously injected ¹¹¹In-BiotinDOTA (ST2210) in the site previously treated with modified avidin underlines that tissue-bound oxidized avidin retains its biotin binding capacity in vivo. The results presented here indicate that oxidized avidin could be employed to create a stable artificial receptor in diseased tissues for the targeting of biotinylated therapeutics.     

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.     

The biotin requirement of HeLa cells

We have examined the effect of alterations in the biotin content of the medium on the growth, viability, biotin content, and the activities of biotin-dependent and biotin-independent enzymes of the HeLa cells. The inclusion in the growth medium of avidin, which almost irreversibly binds with biotin (K_d, 10^?15 M), results in an increase in cellular biotin content and biotin enzyme activity over that seen when the cells are grown in a biotin-depleted medium. The addition of avidin-bound biotin to the growth medium led to a forty-fold increase in cellular biotin when compared to the inclusion of an equivalent amount of free biotin in the medium. HeLa cells are able to internalize avidin-bound biotin. Biotin is released from this complex to function as the prosthetic group of biotin enzymes. HeLa cells do have a nutritional requirement for biotin.     

Biological evaluation of a cytotoxic 2-substituted benzimidazole copper(II) complex: DNA damage,antiproliferation and apoptotic induction activity in human cervical cancer cells

Exploring novel chemotherapeutic agents is a great challenge in cancer medicine. To that end, 2-substituted benzimidazole copper(II) complex, [Cu(BMA)Cl₂]·(CH₃OH) (1) [BMA = N,N′-bis(benzimidazol-2-yl-methyl)amine], was synthesized and its cytotoxicity was characterized. The interaction between complex 1 and calf thymus DNA was detected by spectroscopy methods. The binding constant (K _b = 1.24 × 10⁴ M^?1) and the apparent binding constant (K _app = 6.67 × 10⁶ M^?1) of 1 indicated its moderate DNA affinity. Complex 1 induced single strand breaks of pUC19 plasmid DNA in the presence of H₂O₂ through an oxidative pathway. Cytotoxicity studies proved that complex 1 could inhibit the proliferation of human cervical carcinoma cell line HeLa in both time- and dose-dependent manners. The results of nuclei staining by Hoechst 33342 and alkaline single-cell gel electrophoresis proved that complex 1 caused cellular DNA damage in HeLa cells. Furthermore, treatment of HeLa cells with 1 resulted in S-phase arrest, loss of mitochondrial potential, and up-regulation of caspase-3 and -9 in HeLa cells, suggesting that complex 1 was capable of inducing apoptosis in cancer cells through the intrinsic mitochondrial pathway.     

Synthesis, cellular uptake and HIV-1 Tat-dependent trans-activation inhibition activity of oligonucleotide analogues disulphide-conjugated to cell-penetrating peptides

Oligonucleotides composed of 2′-O-methyl and locked nucleic acid residues complementary to HIV-1 trans-activation responsive element TAR block Tat-dependent trans-activation in a HeLa cell assay when delivered by cationic lipids. We describe an improved procedure for synthesis and purification under highly denaturing conditions of 5′-disulphide-linked conjugates of 3′-fluorescein labelled oligonucleotides with a range of cell-penetrating peptides and investigate their abilities to enter HeLa cells and block trans-activation. Free uptake of 12mer OMe/LNA oligonucleotide conjugates to Tat (48–58), Penetratin and R₉F₂ was observed in cytosolic compartments of HeLa cells. Uptake of the Tat conjugate was enhanced by N-terminal addition of four Lys or Arg residues or a second Tat peptide. None of the conjugates entered the nucleus or inhibited trans-activation when freely delivered, but inhibition was obtained in the presence of cationic lipids. Nuclear exclusion was seen for free delivery of Tat (48–58), Penetratin and R₉ conjugates of 16mer phosphorothioate OMe oligonucleotide. Uptake into human fibroblast cytosolic compartments was seen for Tat, Penetratin, R₉F₂ and Transportan conjugates. Large enhancements of HeLa cell uptake into cytosolic compartments were seen when free Tat peptide was added to Tat conjugate of 12mer OMe/LNA oligonucleotide or Penetratin peptide to Penetratin conjugate of the same oligonucleotide.     

Regulation of high- and low-affinity epidermal growth factor receptors by glucocorticoids

It is reported that receptors for epidermal growth factor (EGF) in HeLa S₃ cells exist in two forms, which differ in both affinity and capacity. Both the number of receptors and their distribution into low- and high-affinity forms are modulated by glucocorticoids. Scatchard analysis of saturation binding assays performed at 0 °C indicates that there is a low-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 1.5}\text{nm}$), which contains approximately 6 × 10⁴ binding sites per cell, and a second, high-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 0.16}\text{nm}$) containing approximately 5 × 10³ binding sites per cell. Exposure of HeLa S₃ cells to 10^?7m dexamethasone for 24 h increased EGF binding to whole cells by increasing the numbers of low- and high-affinity receptors by 20 and 114%, respectively. The increase in EGF binding depends upon the dose of dexamethasone, being raised from 10^?11 to 10^?6m. EGF binding is half-maximal near 2–4 × 10^?9m, a concentration equal to the K_d of dexamethasone for the glucocorticoid receptor in these cells. The increase in EGF binding is specific for glucocorticoids, occurring when the HeLa S₃ cells are exposed to 10^?7m cortisol or dexamethasone for 24 h, but not when the cells are similarly treated with testosterone, 5α-dihydroxytestosterone, 17β-estradiol, or progesterone. The effect on EGF binding appears to be biphasic; the initial rapid increase occurs between 8 and 12 h, is blocked by both 10^?6m cyclohexamide and 0.1 μg/ml actinomycin D, and is followed by a more gradual increase thereafter. These data indicate that glucocorticoids are able to regulate both the number of EGF receptors and their distribution into high- and low-affinity components. Press, Inc.     

The biotin requirement of human fibroblasts in culture

We have examined the effect of biotin deficiency on the growth, viability, biotin content, and the activities of biotin-dependent and biotin-independent enzymes of human fibroblasts. There was a significant decrease in viability of the biotin-deficient cells even when the medium contained serum lipids. Propionyl CoA carboxylase activity reflected the decreased biotin content of the cells whereas alkaline phosphatase activity was not altered. The inclusion of avidin bound biotin in the growth medium resulted in an increase in biotin content as well as propionyl CoA carboxylase activity over that seen when free biotin was included in the medium. The cells appeared to bind and internalize the avidin-biotin complex by adsorptive pinocytosis. These findings are similar to those demonstrated using HeLa cells.     

The malonate decarboxylase enzyme system of Malonomonas rubra: evidence for the cytoplasmic location of the biotin-containing component

Malonate decarboxylase of Malonomonas rubra is a complex enzyme system involving cytoplasmic and membrane-bound components. One of these is a biotin-containing protein of M_r 120'000, the location of which in the cytoplasm was deduced from the following criteria: (i) If the cytoplasm was incubated with avidin and the malonate decarboxylase subsequently completed with the membrane fraction the decarboxylase activity was abolished. The corresponding incubation of the membrane with avidin, however, was without effect. (ii) Western blot analysis identified the single biotin-containing polypeptide of M_r 120'000 within the cytoplasm. (iii) Transmission electron micrographs of immuno-gold labeled M. rubra cells clearly showed the location of the biotinyl protein within the cytoplasm, whereas the same procedure with Propionigenium modestum cells indicated the location of the biotin enzyme methylmalonyl-CoA decarboxylase in the cell membrane. The biotin-containing protein of the M. rubra malonate decarboxylase enzyme system was not retained by monomeric avidin-Sepharose columns but could be isolated with this column in a catalytically inactive form in the presence of detergents. If the high binding affinity of tetrameric avidin towards biotin was reduced by destructing part of the tryptophan residues by irradiation or oxidation with periodate, the inhibition of malonate decarboxylase by the modified avidin was partially reversed with an excess of biotin. Attempts to purify the biotin protein in its catalytically active state using modified avidin columns were without success.     

Inhibition of NADH oxidase activity and growth of HeLa cells by the antitumor sulfonylurea,N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY181984) and response to epidermal growth factor

Right side-out plasma membrane vesicles isolated from HeLa cells exhibited an NADH oxidase activity at their external surfaces that was inhibited by the antitumor sulfonylurea, N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY181984). Intact HeLa cells (fresh or frozen) also exhibited an NADH oxidase activity at the external cell surface. The inhibition of this activity by LY181984 was enhanced by the addition of epidermal growth factor (EGF). The order of addition was critical. It was necessary that the LY181984 be followed by the EGF. If the EGF was administered first, the response to LY181984 was unaffected by EGF. Binding of [³H]LY181984 to HeLa cells also was enhanced by EGF. Growth experiments with HeLa cells revealed a similar pattern of response to EGF. The EC₅₀ of growth inhibition of LY181984 was about 100 μM. However, if the LY181984 was followed by addition of 10 nM EGF, the EC₅₀ for LY181984 was reduced to about 30 nM which now approximated the previously determined K_d of [³H]LY181984 binding of 30 nM and the EC₅₀ of 30 nM for inhibition of NADH oxidase activity by LY181984 by isolated vesicles of plasma membranes. The tumor-inactive sulfonylurea N-(methylphenylsulfonyl-N′-(phenyl)urea (LY181985) was ineffective in the inhibition of NADH oxidation and of growth with HeLa cells either in the presence or absence of EGF.     

Specific interactions between adenosine and streptavidin/avidin

The screening of ligands against proteins plays important role in drug discovery and biological research. Using a dye labelled Streptavidin binding aptamer (SBA) as a competitive reporter probe, we found that adenosine bound to streptavidin specifically. Fluorescence spectral analysis showed that adenosine bound to both avidin and streptavidin with the K_ds in the range of 0.1–0.2 mM, and these bindings can be blocked by biotin. Although streptavidin and avidin are well-known and widely used in bioanalysis, their biological role is still a riddle so far. Since adenosine is a ubiquitous physiological regulator present in cells, our finding provides new clues for the understanding of the functions of both proteins.     

Ion selective electrode estimation of avidin and biotin using a lysozyme label

A method for the homogeneous estimation of the biotin binding protein, avidin, by use of an enzyme label is described. As in homogeneous enzyme immunoassay, where no separation step is employed, the activity of a biotin-lysozyme conjugate is inhibited by the binding of avidin, instead of an immunoagent. Biotin concentration can also be related to conjugate activity after sequential saturation of a known amount of avidin by the biotin sample and the biotin-lysozyme conjugate. Conjugate activity is followed potentiometrically by the release of trimethylphenylammonium ion from loaded Micrococcus lysodeikticus cells or turbidimetrically using a M. lysodeikticus cell suspension.     

Magnetic resonance imaging visualization of targeted cells by the internalization of supramolecular adducts formed between avidin and biotinylated Gd3+ chelates

The high binding affinity between avidin and biotin has been exploited to develop a procedure for magnetic resonance imaging (MRI) visualization of target cells. SHIN3 and PANC1 tumor cell lines have been used as target cells because they possess on their membranes galactosyl receptors able to bind avidin molecules. Avidin–Gd chelate adducts have been built by using two Gd complexes containing one (Gd-I) and two (Gd-II) biotin residues, respectively. The relaxivities of such supramolecular adducts are significantly higher than those shown by free Gd-I and Gd-II. There is evidence of the occurrence of multilayered adducts in which the bis-biotinylated Gd³⁺ complex acts as a bridge between adjacent avidin molecules. MRI differentiation of labeled versus unlabeled cells has been attained when approximately 6×10⁸ Gd units were internalized in each cell. Furthermore, there is a marked decrease in the measured intracellular T₁ relaxivity as the number of internalized Gd complexes increases, probably owing to too short relaxation times of endosomic water protons with respect to their diffusion lifetime.     

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.     

Dissociative binding of alpha- and beta-sulphoxides of biotinylamidoethyl-3-(4-hydroxy-3-[125I]iodophenyl)propionamide to avidin.

Previously we reported the dissociative binding of biotinylamidoethyl-3-(4-hydroxy-3-[125I]iodophenyl)propionamide to avidin [Garlick & Giese (1988) J. Biol. Chem. 263, 210-215]. In the present paper we report the corresponding binding of the alpha- and beta-sulphoxides of this parent compound to avidin. The 1:1 complex (obtained with avidin in excess) of the alpha-sulphoxide derivative with avidin has a dissociation half-life (t1/2) of 25 days, only 1.6 times as fast as the parent compound (t1/2 41 days). However, the corresponding beta-sulphoxide dissociates 446 times faster (t1/2 0.092 day) than the parent compound, this apparently being due to a steric effect. The alpha-sulphoxide is attractive as a tracer reagent to facilitate studies and applications of the avidin-biotin system.     

Specific binding of progesterone to the cell surface and its role in the meiotic divisions in Rana oocytes

Progesterone is believed to act at the cell surface to induce the resumption of the meiotic divisions in amphibian oocytes. Analysis of [³H]- and [¹⁴C]progesterone uptake and exchange by the plasma-vitelline membrane complex, nucleus and cytoplasm of the isolated Rana oocyte indicates that progesterone uptake by the plasma membrane is saturable, specific and temperature-dependent, and has a slow off-rate. Estradiol (a noninducer) did not compete with progesterone, whereas testosterone (an inducer) blocked progesterone uptake by the membrane complex. Scatchard-type plots indicate an apparent K_d of 5.1·10⁻⁷ M over the [progesterone]_o range of 0.01–1.0 μM with maximum binding at about 70 fmol per oocyte. Membrane uptake at higher [progesterone]_o (2–40 μM) indicates apparent cooperative binding, with saturation up to 10 pmol per oocyte. Cytoplasmic uptake was apparently nonspecific and less temperature-dependent than membrane uptake and steroid concentrations (progesterone and pregnanediones) exceeded water solubility by 30–60 min. Nuclear uptake was saturable and specific but uptake was independent of temperature. A comparison of membrane binding and a physiological response (nuclear breakdown) indicated only about 10% of the membrane sites need be filled to initiate a 50% response.     

Binding of methylmercury(II) by HeLa S3 suspension-culture cells: Intracellular methylmercury levels and their effect on DNA replication and protein synthesis

HeLa S3 cells were exposed to varied concentrations of methylmercury over varied periods of time and its binding by the cells was studied using ²⁰³Hg-labeled methylmercuric chloride as radioactive marker. Also studied was the effect of cell-bound methylmercury on DNA replication and protein synthesis and on the growth rate of the cells. The results show that methylmercury binding is a rapid process, with much of the organomercurial bound within the the first 60 min of incubation, and that considerable quantities of organic mercury become affixed to the cells. The amounts of bound methylmercury, [CH₃Hg(II)]_bound, given in mol/cell, range from 2 × 10^?16 (at 1 h of incubation and at 1 μM CH₃Hg(II) in the medium) to almost 4 × 10^?14 (at 24 h of incubation and at 100 μM CH₃Hg(II) in the medium). A [CH₃Hg(II)]_bound value of about 30 × 10^?16 mol/cell appears to be the threshold below which cells display a normal growth pattern and below which metabolic events such as DNA replication or protein synthesis are affected only to a minor degree but above which major changes in cell metabolism and cell growth take place. Methylmercury binding by the cells is tight so that only 20% of the bound material is released from the cells over a 3-h incubation period when the cells are placed into fresh, methylmercury-free growth medium. Analysis of the binding data in terms of binding to identical and completely independent sites yields an association constant K of 7.92 × 10⁴ l/mol and for the maximum concentration of cellular binding sites the value 2.40 × 10^?14 mol/cell or 1.45 × 10¹⁰ sites/cell. Evidence is presented which shows that cellular sulfhydryl groups do not suffice to provide all the sites taken up by methylmercury and that binding, in all likelihood, involves basic nitrogen, too. The levels of cell-bound methylmercury are such that binding to HeLa DNA and HeLa chromatin, for instance, can readily take place. Methylmercury binding data obtained by using the technique of particle-induced X-ray emission (PIXE) are in good agreement with the data obtained via isotope dilution.     

Study on potential antitumor mechanism of a novel Schiff base copper(II) complex: synthesis, crystal structure, DNA binding, cytotoxicity and apoptosis induction activity

A new cytotoxic copper(II) complex with Schiff base ligand [Cu^II(5-Cl-pap)(OAc)(H₂O)]·2H₂O (1) (5-Cl-pap = N-2-pyridiylmethylidene-2-hydroxy-5-chloro-phenylamine), was synthesized and structurally characterized by X-ray diffraction. Single-crystal analysis revealed that the copper atom shows a 4 + 1 pyramidal coordination, a water oxygen appears in the apical position, and three of the basal positions are occupied by the NNO tridentate ligand and the fourth by an acetate oxygen. The interaction of Schiff base copper(II) complex 1 with DNA was investigated by UV-visible spectra, fluorescence spectra and agarose gel electrophoresis. The apparent binding constant (K_app) value of 6.40 × 10⁵ M^− 1 for 1 with DNA suggests moderate intercalative binding mode. This copper(II) complex displayed efficient oxidative cleavage of supercoiled DNA, which might indicate that the underlying mechanism involve hydroxyl radical, singlet oxygen-like species, and hydrogen peroxide as reactive oxygen species. In addition, our present work showed the antitumor effect of 1 on cell cycle and apoptosis. Flow cytometric analysis revealed that HeLa cells were arrested in the S phase after treatment with 1. Fluorescence microscopic observation indicated that complex 1 can induce apoptosis of HeLa cells, whose process was mediated by intrinsic mitochondrial apoptotic pathway owing to the activation of caspase-9 and caspase-3.     

A Power-Law Dependence of Bacterial Invasion on Mammalian Host Receptors

Pathogenic bacteria such as Listeria and Yersinia gain initial entry by binding to host target cells and stimulating their internalization. Bacterial uptake entails successive, increasingly strong associations between receptors on the surface of bacteria and hosts. Even with genetically identical cells grown in the same environment, there are vast differences in the number of bacteria entering any given cell. To gain insight into this variability, we examined uptake dynamics of Escherichia coli engineered to express the invasin surface receptor from Yersinia, which enables uptake via mammalian host β₁-integrins. Surprisingly, we found that the uptake probability of a single bacterium follows a simple power-law dependence on the concentration of integrins. Furthermore, the value of a power-law parameter depends on the particular host-bacterium pair but not on bacterial concentration. This power-law captures the complex, variable processes underlying bacterial invasion while also enabling differentiation of cell lines.