Addition Of Avidin Research Articles

Artificial tethering of LC3 or p62 to organelles is not sufficient to trigger autophagy

The retention using selective hooks (RUSH) system allows to retain a target protein fused to green fluorescent protein (GFP) and a streptavidin-binding peptide (SBP) due to the interaction with a molar excess of streptavidin molecules (“hooks”) targeted to selected subcellular compartments. Supplementation of biotin competitively disrupts the interaction between the SBP moiety and streptavidin, liberating the chimeric target protein from its hooks, while addition of avidin causes the removal of biotin from the system and reestablishes the interaction. Based on this principle, we engineered two chimeric proteins involved in autophagy, namely microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B, best known as LC3) and sequestosome-1 (SQSTM1, best known as p62) to move them as SBP–GFP–LC3 and p62–SBP–GFP at will between the cytosol and two different organelles, the endoplasmic reticulum (ER) and the Golgi apparatus. Although both proteins were functional in thus far that SBP–GFP–LC3 and p62–SBP–GFP could recruit their endogenous binding partners, p62 and LC3, respectively, their enforced relocation to the ER or Golgi failed to induce organelle-specific autophagy. Hence, artificial tethering of LC3 or p62 to the surface of the ER and the Golgi is not sufficient to trigger autophagy.

Acoustic enhancement with fundamental imaging and CPS imaging: comparison between nano-scale PFOB lipidic particles and C3F8 lipidic microbubbles,a vitro experimental study

Objective To prepare two kinds of nano-scale ultrasound contrast agents, perfluorooctyl bromide(PFOB) lipidic particles and perfluoropropane(C3F8) lipidic microbubbles, and compare their characteristics of acoustic enhancement with fundamental imaging and contrast pulse sequence(CPS) imaging in vitro. Methods Physico-chemical properties of PFOB lipidic particles and C3F8 lipidic microbubbles were measured, including morphous, size distribution, Zeta potential, concentration and stability. PFOB lipidic particles with and without biotin, C3F8 lipidic microbubbles with and without biotin were prepared, respectively. Size distribution of four contrast agents were measured, before and after adding avidin. Then, four kinds of ultrasound contrast agents were imaged with both fundamental and CPS imaging before and after avidin was added. Signal intensities were compared using METLAB software. Results ①Addition of avidin had no effect on particle diameters of control lipidic particles/microbubbles. However, the particle diameters of biotinylated lipidic particles and microbubbles were both enlarged with the addition of avidin (P<0.05). ②With fundamental imaging, neither control nor biotinylated PFOB particles were visible before adding avidin. After avidin was added, significant signal enhancement from biotinylated PFOB particles was detected (P<0.05); while control PFOB particles were still unvisible.However, with fundamental imaging, both control and biotinylated C3F8 microbubbles manifested ultrasonic backscatter before and after adding avidin, without significant difference between their signal intensity. ③CPS imaging couldn′t detect nonlinear signal from biotinylated PFOB particles neither before nor after the addition of avidin. However, CPS imaging could detect nonlinear signal from biotinylated C3F8 microbubbles both before and after adding avidin, without significant difference in their signal intensity. Conclusions Compared to C3F8 microbubbles, PFOB particles have better particle size controllability, and higher signal-to-noise (target to nontarget signal) ratio, thus are more suitable for targeted contrast ultrasound imaging. Key words: Ultrasonography; Microbubbles; Liposomes; Perfluorooctyl bromide

TU‐G‐134‐04: Multifunctional Molecular Probes for Targeting and Imaging of Epidermal Growth Factor Receptors in Vivo

Purpose: To demonstrate the epidermal growth factor receptor (EGFR) targeting specificity in‐vivo of a multifunctional molecular probe by combining receptor‐targeting monoclonal antibody (mAb) with the positive contrast agent Gadolinium (Gd) in dendrimer nanoclusters. Methods: The molecular probe comprised two functional subunits: anti‐EGFR antibodies for tumor targeting and dendrimer nanoclusters (DNCs) loaded with Gd for MRI contrast. DNCs were formed by crosslinking G5 dendrimers using Bis‐N‐succinimidyl‐(pentaethylene glycol) ester and sulfo‐NHS‐LC‐biotin. DiethyleneTriaminePentaacetic Acid (DTPA) anhydride was used to bind Gd to the DNC in alkaline conditions. Biotinylated anti‐EGFR mAb(C225) was mixed with the DNC, followed by the addition of avidin, a cross‐linker that facilitates probe synthesis. The probe was injected through the tail vein into nude mice that were subcutaneously implanted with human head and neck cancer (TU159) xenografts. A 3‐dimensional FLASH‐MRI time course study was performed on a 7T scanner, such that volumetric images were acquired every minute in the first 5 min after the injection, every 5 min in the first hour, and after 4 hours. Region of interest (ROI) analysis was performed using ImageJ. Results: An increase in image contrast was observed in the ROI corresponding to the targeted tumor following the tail vein injection of the probe. The time course study showed that the image contrast (IC) at the target region increased substantially in the first 5 min after probe injection and was stable through the first 240 min. In a control experiment, the IC increase was observed on the mouse with the same injected dose of gadodiamine; however in this case, IC subsided after the first 30 min. Conclusion: The molecular nanoprobe is able to 1) selectively target the EGF receptors in vivo and 2) allow drug uptake to be imaged while using less toxic probe doses.

An in vitro comparative study of the physical and imaging enhancement characteristics between two nano-scale ultrasound contrast agents: PFOB lipid particles and C3F8 lipid microbubbles

Objective To prepare two nano-scale ultrasound contrast agents,lipid particles and microbubbles,and to compare their physical and imaging enhancement characteristics.Methods Lipid nanoparticles were prepared with phospholipid and liquid perfluorocarbon (PFOB).Lipid microbubbles were prepared with phospholipid and perfluoropropane(C3F8).Physical and chemical properties of the two ultrasound contrast agents were measured,including morphology,size,zeta potential,concentration and stability.Both lipid nanoparticles and microbubbles with/without biotin were imaged using a 12.0 MHz linear-array transducer with/without avidin.The imaging data were analyzed by using Matlab software.Comparative data between the two groups were statistically analyzed by two-sample t test.Repeated measures analysis of variance was used among the observations at multiple time points within the same sample and Bonferroni method was used for post hoc analysis.Comparison of the signal intensity with and without avidin of the same sample was performed by paired t test.Results (1) Microscopically,both PFOB lipid particles and C3F8 microbubbles were in form of spheres.Their particle diameters were (152.30 ± 35.99) nm and (774.59 ± 108.59) nm,respectively (t =-24.327,P ＜ 0.001).Their zeta potential was (-40.90 ±6.51) mV and (-14.80 ± 3.97) mV,respectively (t =-15.308,P ＜ 0.001).(2) Concentration and size of PFOB lipid particles remained stable during the whole observation period (C0h =(2.28 ± 0.64) ×1011/ml,C1w =(2.06 ±0.53) × 1011/ml; D0h =(152.30 ±35.99) nm,D1 w =(178.80 ±63.07) nm).Concentrations of lipid microbubbles were stable within 12 h (C0h =(4.08 ± 0.96) × 1010/ml,C12h =(3.25 ± 1.02) × 1010/ml) ; and then reduced significantly after 24 h,2 d,4 d and 1 week (C24h:(2.28 ±0.73) ×1010/ml; C2d:(1.56±0.54) ×1010/ml; C4d:(1.03±0.37) ×1010/ml; C1w:(0.74 ±0.24) ×1010/ml ; F =78.515,P ＜ 0.01).Diameters of the particles remained stable within 2 d (D0h =(774.59 ±108.59) nm,D2d =(1020.68 ± 223.64) nm) ; and then increased after 4 d and 1 week (D4d:(1391.67 ±268.65) nm; D1w:(1532.41 ± 326.25) nm,respectively,F =50.772,P ＜ 0.01).(3) Neither control nor biotinylated PFOB particles were visible without avidin.With the addition of avidin,significantly enhanced images were generated by biotinylated PFOB particles (2.18 ±0.71,82.19 ± 15.68,t =-15.698,P ＜ 0.001) but not with the control PFOB particles.Both control and biotinylated C3F8 microbubbles showed no significant difference in their signal intensity with/without avidin (29.16 ± 6.84 and 31.34 ±7.03,33.69±8.24 and28.75±7.18,t=0.653,-1.393,bothP＞0.05).Conclusion Compared to C3F8 microbubbles,PFOB particles have smaller size,greater stability,higher SNR ratio and better imaging enhancement characteristics,therefore,more suitable for use as the target contrast agent for ultrasound imaging. Key words: Contrast media; Ultrasonography ; Lipid nanoparticles ; Lipid microbubbles ; Comparative study

Photoreactive nanotool for cell aggregation and immobilization

We developed a new method to prepare aggregates of specific cells and immobilize cells on a substrate with specific shapes by using a synthetic multifunctional tool, which consisted of a cell adhesive Arg-Gly-Asp (RGD) sequence, a photoreactive phenyl azido group and a biotin group. This chemical nanotool, RGD-2-(6-[biotinamido]-2-(p-azidobenzamido)-hexaneamido)ethyl-1,3′-dithio-proprionate (RGD-BED) was added to human umbilical vein endothelial cells to bind to receptors via the ligand–receptor interaction. Next a photoimmobilization of the binding RGD-BED was carried out by UV irradiation to covalently couple a phenyl azido moiety of RGD-BED with the neighboring site of the integrin receptor. We found that not only the migration distance of RGD-BED immobilized cells was diminished, but also the cell morphology was fixed on the substrate due to the blocking of integrin receptors by RGD-BED. In contrast, the addition of biocytin-containing polymers, poly(N-methacryloyloxy biocytin-co-dimethylacrylamide), and avidin to the RGD-BED-immobilized cells led to restore cellular migration behavior, probably arising from the increase in the rigidity of the environment surrounding the cells. Furthermore, by the addition of avidin to the RGD-BED-immobilized cells, three-dimensional cell aggregates were formed due to the cross-linking of the biotin moieties of RGD-BED. These results show that RGD-BED is a potential nanotool not only to label and collect targeted cells by the formation of cell aggregates but also to suppress mobility and morphologies of specific cells to be applicable for medical treatments.

Immobilization of double functionalized carbon nanotubes on glassy carbon electrodes for the electrochemical sensing of the biotin–avidin affinity

Multi-walled carbon nanotubes (MWCNTs) double functionalized with redox-active ferrocene and biotin ( Fc–Biot-MWCNTs) were synthesized and used for the electrochemical detection of avidin. After dispersion in perfluorosulfonated polymer Nafion and immobilization on the electrode surfaces, the cyclic voltammetry response of the modified electrodes showed in aqueous medium a quasi-reversible one-electron system at 0.46 V vs. SCE, assigned to the bound ferrocene/ferrocenium redox couple. Upon the addition of avidin in the range 0.9–20 nM, a stepwise decrease of both anodic and cathodic peak currents ascribed to the ferrocene was observed. These electrochemical changes are specifically due to the formation of biotin–avidin complex and are explained by complexation-induced modifications in the environment of covalently bound ferrocene.

Immobilization of Peroxidase on SiO2 Surfaces with the Help of a Dendronized Polymer and the Avidin‐Biotin System

Horseradish peroxidase (HRP) is immobilized in three easy steps on SiO(2) surfaces with the help of a polycationic second generation dendronized polymer (denpol) and the biotin-avidin system. This stepwise immobilization process is monitored and quantitatively analyzed with the transmission interferometric adsorption sensor. Partially biotinylated denpol is first adsorbed onto SiO(2) , followed by addition of avidin and then of biotinylated HRP. Denpols in their molecular structure combine properties of polymers as well as dendrimers which are found to be of clear advantage for this type of non-covalent enzyme immobilization. With respect to the reproducibility of the adsorption process and with respect to the stability of the adsorbed polymer layer, the denpol is superior to α-poly-D-lysine which is used as a reference polymer. Furthermore, HRP immobilized with the denpol on commercial glass slides remains considerably more active upon storage as compared to HRP immobilized with the help of α-poly-D-lysine with a similar number of repeating units. The ease of the denpol-mediated HRP immobilization and the high stability of the immobilized enzyme are promising for bioanalytical applications.

Nanoparticle Agglutination: Acceleration of Aggregation Rates and Broadening of the Analyte Concentration Range Using Mixtures of Various-Sized Nanoparticles

SiO(2) particles of various sizes were prepared and surface modified with biotin-chain-end-functionalized poly(ethylene glycol). Dispersions of these particles were prepared, and their aggregation was induced upon the addition of avidin. The aggregate size and growth rate were monitored by DLS analysis, and SEM and TEM images of freeze-dried samples of the aggregate solutions were used to confirm the DLS data and to image the aggregate size and dimension. A linear correspondence between apparent diameter and time was observed, and both the 20 and 300 nm particles aggregated at slower rates than for the 40 nm particles. These observations were attributed to differences in the average functionality of the systems and the different initial concentrations of particles and avidin. The observed aggregation rates of binary combinations of the three particle sizes (i.e., 20 + 40 nm or 40 + 300 nm) were faster than those of the single-sized mixtures. These results were attributed to the faster flux of smaller particles toward larger particles in the mixture solutions as well as to the potentially larger number of productive collisions possible between mixtures of small particles and large particles versus only similarly sized particles. Combinations of the three sizes of particles were studied to find an empirical optimum formulation for generating large aggregates on a short time scale and over a wide range of analyte concentrations.

Synthesis and properties of a novel water-soluble anionic polyfluorenes for highly sensitive biosensors

A new anionic water-soluble polyfluorene—poly(9,9-bis(4′-sulfonatobutyl)fluorene- co- alt-1,4-phenylene) sodium salt (PFS) was synthesized by Suzuki-coupling reaction. The anionic polyfluorene shows bright blue photoluminescence and it was found the photoluminescence could be highly quenched by a bioactive dye (Lucifer yellow cadaverine biotin-X dipotassium salt). PFS photoluminescence quenching only occurs effectively upon addition of avidin. The photoluminescence quenching process is sensitive to even subnanomolar amount of avidin (10 −10 M). The avidin facilitated photoluminescence quenching opens up opportunities of new ultra-high efficiency and rapid response biosensors based on the polyfluorene electrolyte.

Translocation of Histone Proteins Across Lipid Bilayers and Mycoplasma Membranes

We show that the three core histones H2A, H3 and H4 can transverse lipid bilayers of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). In contrast, the histone H2B, although able to bind to the liposomes, fails to penetrate the unilamellar and the multilamellar vesicles. Translocation across the lipid bilayer was determined using biotin-labeled histones and an ELISA-based system. Following incubation with the liposomes, external membrane-bound biotin molecules were neutralized by the addition of avidin. Penetrating biotin-histone conjugates were exposed by Triton treatment of the neutralized liposomes. The intraliposomal biotin-histone conjugates, in contrast to those attached only to the external surface, were attached to the detergent lysed lipid molecules. Thus, biotinylated histone molecules that were exposed only following detergent treatment of the liposomes were considered to be located at the inner leaflet of the lipid bilayers. The penetrating histone molecules failed to mediate translocation of BSA molecules covalently attached to them. Translocation of the core histones, including H2B, was also observed across mycoplasma cell membranes. The extent of this translocation was inversely related to the degree of membrane cholesterol. The addition of cholesterol also reduced the extent of histone penetration into the MLVs. Although able to bind biotinylated histones, human erythrocytes, erythrocyte ghosts and Escherichia coli cells were impermeable to them. Based on the present and previous data histones appear to be characterized by the same features that characterize cell penetrating peptides and proteins (CPPs).

Protein−Ligand Mediated Aggregation of Nanoparticles: A Study of Synthesis and Assembly Mechanism

Two difunctional, asymmetric PEG linkers were synthesized and utilized to prepare water-soluble, biotinylated CdS@SiO2 particles with various surface contents of the biotin groups. Aqueous dispersions of these particles were prepared, and upon addition of avidin, aggregation was observed using DLS, TEM, and SEM analysis. The resulting aggregates were dense but also had fractal shapes. It was shown that single-angle DLS measurements at 90° were sufficiently accurate for quickly measuring the size of the aggregates as it evolved with time. Initial aggregation rates were measured as a function of surface biotin content of the nanoparticles and initial avidin concentration. The initial rates displayed bell-shaped curve responses to variations in the avidin-to-particle ratio and biotin-to-avidin ratio, the shapes of which were attributed to the stoichiometric balance/imbalance of the avidin-to-biotin ratio in the system and to intraparticle and interparticle bridges that could sterically block avidin from reaching some biotin sites. The growth mechanism was postulated to be a step-growth process.

Modulation of the pharmacokinetics of macromolecular contrast material by avidin chase: MRI, optical, and inductively coupled plasma mass spectrometry tracking of triply labeled albumin.

The goal of this work was to develop an MRI method for mapping the clearance of interstitial macromolecular plasma proteins after their extravasation from permeable blood vessels. To that end, a well-defined window of exposure to elevated blood levels was generated by inducing rapid clearance of macromolecular contrast material from the blood. Experimental removal of the intravascular component allowed subsequent tracking of clearance from the interstitial compartment in the absence of further contrast extravasation. The contrast material was based on albumin triply labeled with biotin, fluorescent tag, and GdDTPA, allowing optical, inductively coupled plasma mass spectrometry (ICP-MS) and MRI detection. The biotin tag was used here for in vivo chasing of the contrast material from the blood by intravenous administration of avidin. Upon administration of avidin the contrast material disappeared from the blood vessels and was cleared by the liver and spleen as detected by MRI, fluorescence of blood samples and histological sections, and by ICP-MS. Nonbiotinylated fluorescent albumin was not affected by administration of avidin. Contrast material that extravasated from leaky blood vessels in a VEGF overexpressing tumor, prior to administration of avidin, was not cleared by the addition of avidin and showed continued interstitial convection. Thus, avidin-chase provides an effective tool for in vivo manipulation of the arterial input function by providing experimental control over the rate of clearance of the contrast material from the circulation.

Labeling of biotin with [formula omitted]/ [formula omitted] as a stable in vivo generator system

The aim of this work was to synthesize [ 166 Dy] Dy / 166 Ho -DTPA-Biotin to evaluate its potential as a new radiopharmaceutical for targeted radiotherapy. Dysprosium-166 ( 166 Dy ) was obtained by neutron irradiation of enriched 164 Dy 2 O 3 in a Triga Mark III reactor. The labeling was carried out in aqueous media at pH 8.0 by addition of [ 166 Dy] DyCl 3 to diethylenetriaminepentaacetic-α,ω-bis(biocytinamide) (DTPA-Biotin). Radiochemical purity was determined by high-performance liquid chromatography (HPLC) and TLC. The biological integrity of labeled biotin was studied evaluating its avidity for avidin in an agarose column and by size-exclusion HPLC analysis of the radiolabeled DTPA-Biotin with and without the addition of avidin. Stability studies against dilution were carried out by diluting the radiocomplex solution with saline solution and with human serum at 37 °C for 24 h. The [ 166 Dy] Dy / 166 Ho -labeled biotin was obtained with a 99.1±0.6% radiochemical purity. In vitro studies demonstrated that [ 166 Dy] Dy / 166 Ho -DTPA-Biotin is stable after dilution in saline and in human serum and no translocation of the daughter nucleus occurs subsequent to β − decay of 166 Dy that could produce release of 166 Ho 3+ . Avidity of labeled biotin for avidin was not affected by the labeling procedure. Biodistribution studies in normal mice showed that the [ 166 Dy] Dy / 166 Ho -DTPA-Biotin has a high renal clearance. In conclusion, the radiolabeled biotin prepared in this investigation has adequate properties to work as a stable in vivo generator system for targeted radiotherapy.

Core–Shell Type Polystyrene Latex Possessing Reactive Poly(ethylene glycol) Brushes on the Surface for High Performance Immunodiagnostics

Amphiphilic poly(ethylene glycol)-b-polylactide (PEG/PLA) block macromonomers with an aldehyde group at the PEG chain end and a methacryloyl group at the PLA chain end were quantitatively synthesized by anionic polymerization. PEGylated latex particles were then prepared by dispersion radical copolymerization of the PEG/PLA block macromonomer with styrene in water. PEG/PLA macromonomer itself works as a surfactant to stabilize styrene droplets in the copolymerization process. Acetal groups at the PEG chain end on the latex particle were quantitatively converted to aldehyde groups by an acid treatment. In this way, polystyrene latex particles covered with PEG tethered chains possessing an aldehyde end-group were obtained. The size of the latex was quantitatively controlled ranging in size from 56 nm to 235 nm by the amount of the block macromonomer. The number of aldehyde groups on the latex particle was estimated to be approximately 0.029 molecules nm-2 by electric spin resonance (ESR) using 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) as a probe. Regardless of the size of the latex particles synthesized in this study, the occupied area of 4-amino-TEMPO on the particle was constant. Biotin was then introduced in a distal end of PEG-strands as a model ligand. The biotin-modified PEGylated latex was coagulated in a facile manner by the addition of avidin. From an inhibitory coagulation test using free biotin, it was found that more than 5.8 × 108 times of free biotin was required for the prevention of coagulation. Such a high complex formation ability of the biotin on the latex surface to avidin may be explained by the hypervalency effect. The core-shell type polystyrene latex particles, with reactive PEG brushes thus prepared, show a stable dispersity and a lowered non-specific interaction with proteinous compounds, and may have a promising potential in the diagnostic field.

Vav-2 controls NFAT-dependent transcription inB- but not T-lymphocytes

We show here that Vav-2 is tyrosine phosphorylated following antigen receptor engagement in both B- and T-cells, but potentiates nuclear factor of activated T cells (NFAT)-dependent transcription only in B cells. Vav-2 function requires the N-terminus, as well as functional Dbl homology and SH2 domains. More over, the enhancement of NFAT-dependent transcription by Vav-2 can be inhibited by a number of dominant-negative GTPases. The ability of Vav-2 to potentiate NFAT-dependent transcription correlates with its ability to promote a sustained calcium flux. Thus, Vav-2 augments the calcium signal in B cells but not T cells, and a truncated form of Vav-2 can neither activate NFAT nor augment calcium signaling. The CD19 co-receptor physically interacts with Vav-2 and synergistically enhances Vav-2 phosphorylation induced by the B-cell receptor (BCR). In addition, we found that Vav-2 augments CD19-stimulated NFAT- dependent transcription, as well as transcription from the CD5 enhancer. These data suggest a role for Vav-2 in transducing BCR signals to the transcription factor NFAT and implicate Vav-2 in the integration of BCR and CD19 signaling.

Effect of Avidin on Channel Kinetics of Biotinylated Gramicidin

Membrane protein functioning basically depends on the supramolecular structure of the proteins which can be modulated by specific interactions with external ligands. The effect of a water-soluble protein bearing specific binding sites on the kinetics of ionic channels formed by gramicidin A (gA) in planar bilayer lipid membranes (BLM) has been studied using three independent approaches: (1) sensitized photoinactivation, (2) single-channel, and (3) autocorrelation measurements of current fluctuations. As shown previously [Rokitskaya, T. I., et al. (1996) Biochim. Biophys. Acta 1275, 221], the time course of the flash-induced current decrease in most cases follows a single-exponential decay with an exponential factor (tau) that corresponds to the gA single-channel lifetime. Addition of avidin does not affect tau for gA channels, but causes a dramatic increase in tau for channels formed by gA5XB, a biotinylated analogue of gA. This effect is reversed by addition of an excess of biotin to the bathing solution. The average single-channel duration of gA5XB was about 3.6 s as revealed by single-channel recording of the BLM current. After prolonged incubation with avidin, a long-lasting open state of the gA5XB channel appeared which did not close for more than 10 min. The data on gA5XB photoinactivation kinetics and single-channel measurements were confirmed by analysis of the corresponding power spectra of the current fluctuations obtained in the control, in the presence of avidin, and after the addition of biotin. We infer that avidin produces a deceleration of gA5XB channel kinetics by motional restriction of gA5XB monomers and dimers upon the formation of avidin and gA5XB complexes, which would stabilize the channel state and thus increase the single-channel lifetime.

Optical Absorption Study of the Biotin−Avidin Interaction on Colloidal Silver and Gold Particles

The biotin−avidin reaction is well studied and is often used as a prototypical interaction in the development of immunoassays. In this paper, this reaction is studied on the surface of colloidal silver and gold particles as a first step in the development of a sol-based assay. More specifically, silver and gold colloidal particles were biotinylated by self-assembly of a biotin disulfide molecule, and the reaction of the surface-modified colloidal particles with avidin molecules was followed using optical absorption spectroscopy. The specific interaction of avidin, a tetrameric protein, with biotin leads to cross-linking of the colloidal particles (“flocculation”) and a consequent growth of a long wavelength absorption peak. The degree of flocculation was quantified using a semiempirical flocculation parameter, and the dependence of this parameter on the extent of biotinylation of the colloidal particle surface as well as the concentration of avidin in solution was studied to determine the optimum working conditions of the sol. The silver sol required electrostatic stabilization of the biotin-capped particles through the simultaneous incorporation of a charged bifunctional molecule, 4-carboxythiophenol, in the capping monolayer while the gold sol was stable with biotin capping. Both biotinylated silver and gold sols showed a visible color change on addition of avidin. However, changes in the optical absorption spectra were more marked for the silver sol.

Decarboxylation of 2,3-Dihydroxybenzoate to Catechol Supports Growth of Fermenting Bacteria

Anaerobic enrichment cultures with 2,3-dihydroxybenzoate as sole source of energy and organic carbon yielded slow-growing cultures of fermenting bacteria. A culture derived from a marine inoculum, strain Pe23DHB, consisted of two morphotypes: spirilloid, highly motile bacteria (the predominant type), and very few long, rod-shaped bacteria. The culture was able to grow by decarboxylation of 2,3-dihydroxybenzoate to catechol. The decarboxylating activity was localized mainly in the cytosol; addition of avidin or of sodium salts had no effect on the decarboxylating activity in cell-free extracts. These results suggest that strain Pe23DHB did not conserve energy by a membrane-bound biotin-containing decarboxylase, creating a Na+-gradient across the cytoplasmic membrane. The energy conservation mechanism remains unknown at present.

Identification of a Translocation Intermediate Occupying Functional Protein Import Sites in the Chloroplastic Envelope Membrane

We used complexes of avidin and biotinylated precursors to generate translocation intermediates that occupy functional transport sites and thereby block the transport of other precursor proteins into pea chloroplasts. Cysteine residues of purified precursor to the small subunit of rubisco (prSS) were modified with the biotinylation reagent biotin-1-biotinamido-4-[-4'-(maleimidomethyl)-cyclohexane-ca rboxamido ]butane. Chemically biotinylated prSS was readily imported into chloroplasts. The addition of avidin, however, resulted in the formation of an avidin-biotinylated precursor complex that could not be imported into chloroplasts even when precursors had already engaged the transport apparatus before avidin was added. On fractionation, the avidin-biotinylated precursor complex associated with envelope membranes. Titration of transport sites with avidin-biotinylated precursor complexes revealed that saturation was reached at 2,000 molecules/chloroplast. Even with less than saturating levels of complexes, a sufficient number of translocation sites could be occupied with avidin-precursor complexes so that the import rate of freshly added radiolabeled prSS was reduced by 35%. From these observations we conclude that the trapped intermediates were blocking functional translocation sites. These biotinylated translocation intermediates should be useful in future efforts to purify and characterize the chloroplastic protein import machinery.

Electrochemical Assay of Avidin and Biotin Using a Biotin Derivative Labeled with an Electroactive Compound

A new avidin-biotin assay was developed based on electrochemical detection. The assay is achieved by biotin labeled with an electroactive compound. That is, because avidin-biotin binding makes a labeled biotin electroinactive, avidin-biotin interaction can be detected indirectly by a change in the electrode response of the labeled biotin. Daunomycin was selected as an electroactive labeling reagent for biotin, and labeled biotin was prepared to carry out the electrochemical assay. By the addition of avidin, the electrode response of the labeled biotin decreased linearly over the range of 3×10 -9 and 5×10 -8 mol dm -3 avidin. The relative standard deviation at 3×10 -8 mol dm -3 avidin was about 5% (n=5). On the other hand, a response curve for biotin was obtained by the competition reaction between the labeled biotin and biotin for the limited binding sites of avidin. This method has the advantage that it is not necessary to separate a free labeled biotin from a bound one