24 results on '"Blake,II., Robert C."'
Search Results
2. Yeast Surface Display Platform for Rapid Selection of an Antibody Library via Sequential Counter Antigen Flow Cytometry.
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Ban, Bhupal, Blake II, Robert C., and Blake, Diane A.
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FLOW cytometry , *FLOW meters , *IMMUNOGLOBULINS , *CARRIER proteins , *YEAST , *GENE libraries - Abstract
Yeast surface display techniques have been increasingly employed as a tool for both the discovery and affinity maturation of antibodies. In this study, we describe the use of yeast surface display for the selection and affinity maturation of antibodies targeted to small molecules (haptens). In this approach, we coupled 4 to 15 sequential cycles of error-prone PCR to introduce heterogeneity into the sequence of an 12F6 scFv antibody that binds to chelated uranium; the resulting full-length constructs were combined to create a yeast-displayed scFv-library with high diversity. We also developed a stringent selection technique utilizing fluorescence-activated cell sorting; this was based on sequentially dropping the target antigen concentration, while concomitantly increasing the concentration of potential cross-reactive haptens in subsequent selection cycles. As a proof of the efficacy this approach, we confirmed that the antibodies identified via this approach retained binding to the target antigen (UO22+ complexed to a chelator), while binding with lesser affinity than the parental scFv to a structurally related haptens (the same chelator complexed to other metal ions). As will be described in this report, these scFv variants perform more efficiently in sensor-based assay than the parental 12F6 antibody. Combining the generation of scFv libraries via error-prone PCR with selection of yeast-displayed antibodies by fluorescence activated cell sorting will provide an efficient new method for the isolation of scFvs and other binding proteins with high affinity and specificity. [ABSTRACT FROM AUTHOR]
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- 2022
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3. Expression and purification of the HTLV-1 transforming protein Tax
- Author
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Traina-Dorge Vicki, Blake II Robert C, Korona Boguslawa, Preyan Lynez, Weber Morgan, Carriere Patrick, Evans Whitney, and Shuh Maureen
- Subjects
Immunologic diseases. Allergy ,RC581-607 - Published
- 2011
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4. Community proteomics of a natural microbial biofilm
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Ram, Rachna J., VerBerkmoes, Nathan C., Thelen, Michael P., Tyson, Gene W., Baker, Brett J., Blake, II, Robert C., Shah, Manesh, Hettich, Robert L., and Banfield, Jillian F.
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Microbial mats -- Genetic aspects -- Structure -- Analysis ,Science and technology - Abstract
Using genomic and mass spectrometry-based proteomic methods, we evaluated gene expression, identified key activities, and examined partitioning of metabolic functions in a natural acid mine drainage (AMD) microbial biofilm community. We detected 2033 proteins from the five most abundant species in the biofilm, including 48% of the predicted proteins from the dominant biofilm organism, Leptospirillum group II. Proteins involved in protein refolding and response to oxidative stress appeared to be highly expressed, which suggests that damage to biomolecules is a key challenge for survival. We validated and estimated the relative abundance and cellular localization of 357 unique and 215 conserved novel proteins and determined that one abundant novel protein is a cytochrome central to iron oxidation and AMD formation., Microbial communities play key roles in the Earth's biogeochemical cycles. Our knowledge of the structure and activities in these communities is limited, because analyses of microbial physiology and genetics have [...]
- Published
- 2005
5. Ferrimicrobium acidiphilum Exchanges Electrons With a Platinum Electrode via a Cytochrome With Reduced Absorbance Maxima at 448 and 605 nm.
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Blake II, Robert C., Nautiyal, Amit, Smith, Kayla A., Walton, Noelle N., Pendleton, Brealand, and Wang, Zhe
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PLATINUM electrodes ,STANDARD hydrogen electrode ,OXYGEN electrodes ,LIGHT absorbance ,REDUCTION potential - Abstract
Ferrimicrobium acidiphilum is a Gram-positive member of the Actinobacteria phylum that can respire aerobically or anaerobically with soluble Fe(II) or Fe(III), respectively, in sulfuric acid at pH 1.5. Cyclic voltammetry measurements using intact F. acidiphilum at pH 1.5 produced fully reversible voltammograms that were highly reproducible. The maximum current observed with the anodic peak was considerably less than was the maximum current observed with the cathodic peak. This difference was attributed to the competition between the platinum electrode and the soluble oxygen for the available electrons that were introduced by the cathodic wave into this facultative aerobic organism. The standard reduction potential of the intact organism was determined to be 786 mV vs. the standard hydrogen electrode, slightly more positive than that of 735 mV that was determined for soluble iron at pH 1.5 using the same apparatus. Chronocoulometry measurements conducted at different cell densities revealed that the intact organism remained in close proximity to the working electrode during the measurement, whereas soluble ionic iron did not. When the cyclic voltammetry of intact F. acidiphilum was monitored using an integrating cavity absorption meter, the only small changes in absorbance that were detected were consistent with the participation of a cellular cytochrome with reduced absorbance peaks at 448 and 605 nm. The cytochrome that participated in the exchange of electrons between the intact organism and extracellular solid electrodes like platinum was the same cytochrome whose oxidation was previously shown to be rate-limiting when the organism respired aerobically on extracellular soluble iron. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
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6. Homogeneous Cytochrome 579 Is an Octamer That Reacts Too Slowly With Soluble Iron to Be the Initial Iron Oxidase in the Respiratory Chain of Leptospirillum ferriphilum.
- Author
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Blake II, Robert C., Shively, John E., Timkovich, Russell, and White III, Richard Allen
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CYTOCHROME c ,AMINO acid sequence ,IRON ,IRON oxidation ,LIGHT scattering ,MASS spectrometry - Abstract
The exact role that cytochrome 579 plays in the aerobic iron respiratory chain of Leptospirillum ferriphilum is unclear. This paper presents genomic, structural, and kinetic data on the cytochrome 579 purified from cell-free extracts of L. ferriphilum cultured on soluble iron. Electrospray mass spectrometry of electrophoretically homogeneous cytochrome 579 yielded two principal peaks at 16,015 and 16,141 Daltons. N-terminal amino acid sequencing of the purified protein yielded data that were used to determine the following: there are seven homologs of cytochrome 579; each homolog possesses the CXXCH heme-binding motif found in c -type cytochromes; each of the seven sequenced strains of L. ferriphilum expresses only two of the seven homologs of the cytochrome; and each homolog contains an N-terminal signal peptide that directs the mature protein to an extra-cytoplasmic location. Static light scattering and macroion mobility measurements on native cytochrome 579 yielded masses of 125 and 135 kDaltons, respectively. The reduced alkaline pyridine hemochromogen spectrum of the purified cytochrome had an alpha absorbance maximum at 567 nm, a property not exhibited by any known heme group. The iron-dependent reduction and oxidation of the octameric cytochrome exhibited positively cooperative kinetic behavior with apparent Hill coefficients of 5.0 and 3.7, respectively, when the purified protein was mixed with mM concentrations of soluble iron. Consequently, the extrapolated rates of reduction at sub-mM iron concentrations were far too slow for cytochrome 579 to be the initial iron oxidase in the aerobic respiratory chain of L. ferriphilum. Rather, these observations support the hypothesis that the acid-stable cytochrome 579 is a periplasmic conduit of electrons from initial iron oxidation in the outer membrane of this Gram-negative bacterium to a terminal oxidase in the plasma membrane. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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7. Uranium (VI) detection in groundwater using a gold nanoparticle/paper-based lateral fow device
- Author
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Agencia Estatal de Investigación (España), Ministerio de Economía, Industria y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), European Commission, Ministerio de Economía y Competitividad (España), National Science Foundation (US), Generalitat de Catalunya, Quesada-González, Daniel, Jairo, Grace A., Blake II, Robert C., Blake, Diane A., Merkoçi, Arben, Agencia Estatal de Investigación (España), Ministerio de Economía, Industria y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), European Commission, Ministerio de Economía y Competitividad (España), National Science Foundation (US), Generalitat de Catalunya, Quesada-González, Daniel, Jairo, Grace A., Blake II, Robert C., Blake, Diane A., and Merkoçi, Arben
- Abstract
The contamination in groundwater due to the presence of uranium is nowadays a subject of concern due to the severe health problems associated with renal failure, genotoxicity and cancer. The standard methods to detect uranium require time-consuming processes and expensive non-portable equipment, so these measurements are rarely performed in-field, which increases the time until water samples are analysed. Furthermore, the few portable methods available do not allow quantitative analysis and the detection limit is often not low enough to reach the recommendations for drinking water (30 ppb or 126 nM of uranium). For the first time, we propose a portable, fast, inexpensive and sensitive paper-based biosensor able to detect in situ U(VI) in water samples: U(VI) selective gold nanoparticle-based lateral flow strips. Antibody-coated gold nanoparticles are used as labels in the proposed lateral flow system because of their biocompatibility; in addition, these nanoparticles provide high sensitivity due to their intense plasmonic effect. The antibody used in the assay recognizes soluble U(VI) complexed to the chelator, 2,9-dicarboxyl-1,10-phenanthroline (DCP). Because of the small size of the U(VI)-DCP complex, this assay employs a competitive format that reaches a limit of detection of 36.38 nM, lower than the action level (126 nM) established by the World Health Organization and the U.S. Environmental Protection Agency for drinking waters.
- Published
- 2018
8. Oxidation of cytochrome 605 is the rate limiting step when Ferrimicrobium acidiphilum respires aerobically on soluble iron.
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Blake II, Robert C., Guidry, Jessie J., Anthony, Micah D., Ban, Bhupal, Smith, Kayla A., Walton, Noelle N., and Painter, Richard G.
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MICHAELIS-Menten equation , *GENOMICS , *OPTICAL measurements , *IRON oxidation , *IRON ions , *CYTOCHROME c , *COMPARATIVE genomics - Abstract
Proteins that oxidize extracellular substrates in Gram-positive bacteria are poorly understood. Ferrimicrobium acidiphilum is an actinobacterium that respires aerobically on extracellular ferrous ions at pH 1.5. In situ absorbance measurements were conducted on turbid suspensions of intact Fm. acidiphilum using an integrating cavity absorption meter designed for that purpose. Initial velocity kinetic studies monitored the appearance of product ferric ions in the presence of catalytic quantities of cells. Cell-catalyzed iron oxidation obeyed the Michaelis-Menten equation with values for KM and Vmax of 71 µM and 0.29 fmol/min/cell, respectively. Limited-turnover kinetic studies were conducted with higher concentrations of cells to detect and monitor changes in the absorbance properties of cellular redox proteins when the cells were exposed to limited quantities of soluble reduced iron. A single a-type cytochrome with reduced absorbance peaks at 448 and 605 nm was the only redox-active chromophore that was visible as the cells respired aerobically on iron. The reduced cytochrome 605 exhibited mathematical and correlational properties that were consistent with the hypothesis that oxidation of the cytochrome constituted the rate-limiting step in the aerobic respiratory process with a turnover number of 35 ± 2 s-1. Genomic and proteomic analyses showed that Fm. acidiphilum could and did express only two a-type heme copper terminal oxidases. Cytochrome 605 was associated with the terminal oxidase gene that is located between nucleotides 31090 and 33039, inclusive, in the annotated circular genome of this bacterium. IMPORTANCE The identities and functions of proteins involved in aerobic respiration on extracellular ferrous ions at acidic pH are poorly understood in the 4 phyla of Gram-positive eukaryotes and archaea where such activities occur. In situ absorbance measurements were conducted on Fm. acidiphilum as it respired on extracellular iron using an integrating cavity absorption meter that permitted accurate optical measurements in turbid suspensions of the intact bacterium under physiological conditions. The significance of these measurements is that they permitted a direct spectrophotometric examination of the extents and rates of biological electron transfer events in situ under non-invasive physiological conditions without disrupting the complexity of the live cellular environment. One thing is certain: one way to understand how a protein functions in an intact organism is to actually observe that protein as it functions in the intact organism. This paper provides an example of just such an observation. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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9. Chapter Three - In situ absorbance measurements: a new means to study respiratory electron transfer in chemolithotrophic microorganisms.
- Author
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Blake II, Robert C. and White III, Richard A.
- Abstract
Absorbance measurements on intact chemolithotrophic microorganisms that respire aerobically on soluble iron are described that used a novel integrating cavity absorption meter to eliminate the effects of light scattering on the experimental results. Steady state kinetic measurements on ferric iron production by intact cells revealed that the Michaelis Menten equation described the initial rates of product formation for at least 8 different chemolithotrophic microorganisms in 6 phyla distributed equally among the archaea and the Gram negative and Gram positive eubacteria. Cell-monitored turnover measurements during aerobic respiration on soluble iron by the same 12 intact microorganisms revealed six different patterns of iron-dependent absorbance changes, suggesting that there may be at least six different sets of prosthetic groups and biomolecules that can accomplish aerobic respiration on soluble iron. Detailed kinetic studies revealed that the 3-component iron respiratory chain of Acidithiobacillus ferrooxidans functioned as an ensemble with a single macroscopic rate constant when the iron-reduced proteins were oxidized in the presence of excess molecular oxygen. The principal member of this 3-component system was a cupredoxin called rusticyanin that was present in the periplasm of At. ferrooxidans at an approximate concentration of 350 mg/mL, an observation that provides new insights into the crowded environments in the periplasms of Gram negative eubacteria that conduct electrons across their periplasm. The ability to conduct direct spectrophotometric measurements under noninvasive physiological conditions represents a new and powerful approach to examine the rates and extents of biological events in situ without disrupting the complexity of the live cellular environment. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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10. In situ Spectroscopy Reveals that Microorganisms in Different Phyla Use Different Electron Transfer Biomolecules to Respire Aerobically on Soluble Iron
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Blake II, Robert C., primary, Anthony, Micah D., additional, Bates, Jordan D., additional, Hudson, Theresa, additional, Hunter, Kamilya M., additional, King, Brionna J., additional, Landry, Bria L., additional, Lewis, Megan L., additional, and Painter, Richard G., additional
- Published
- 2016
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11. Immunoassays for metal ions
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Blake, Diane A., primary, Blake II, Robert C., additional, Khosraviani, Mehraban, additional, and Pavlov, Andrey R., additional
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- 1998
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12. Multiple Wavelength Anomalous Diffraction (MAD) Crystal Structure of Rusticyanin: a Highly Oxidizing Cupredoxin with Extreme Acid Stability
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Walter, Richard L., primary, Ealick, Steven E., additional, Friedman, Alan M., additional, Blake II, Robert C., additional, Proctor, Peter, additional, and Shoham, Menachem, additional
- Published
- 1996
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13. NMR Solution Structure of Cu(I) Rusticyanin fromThiobacillus ferrooxidans: Structural Basis for the Extreme Acid Stability and Redox Potential
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Botuyan, Maria Victoria, primary, Toy-Palmer, Anna, additional, Chung, John, additional, Blake II, Robert C., additional, Beroza, Paul, additional, Case, David A., additional, and Dyson, H.Jane, additional
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- 1996
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14. Electrospray Ionization&3x2014;Ion Mobility Spectrometry Identified Monoclonal Antibodies that Bind Exclusively to Either the Monomeric or a Dimeric Form of Prostate Specific Antigen.
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Blake, II, Robert C. and Blake, Diane A.
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MONOCLONAL antibodies , *ELECTROSPRAY ionization mass spectrometry , *MACROMOLECULES , *ANTIGENS , *IMMUNOGLOBULIN analysis , *BINDING sites , *PROSTATE-specific antigen - Abstract
Macroion mobility spectrometry was used to distinguish between a monoclonal antibody (clone M612165) that bound exclusively to monomeric prostate specific antigen and a different monoclonal antibody (clone M612166) that bound exclusively to a dimeric form of the antigen that only comprised 6.8% of the total protein. In the presence of excess antigen, the mobility spectrum of M612165 was replaced by a composite spectrum that represented a mixture of antibodies that included either one or two equivalents of the protein antigen. In similar circumstances, the mobility spectrum of M612166 was replaced by a composite spectrum that represented a mixture of antibodies that included either two or four equivalents of the protein antigen. When exposed to either of the two antibodies, the mobility spectrum of the prostate specific antigen showed a concomitant decrease in the monomeric antigen in one case and in the dimeric antigen in the other case. While sensitive kinetic exclusion assays demonstrated large differences in the antigen binding behavior of the two antibodies, these functional studies alone were insufficient to reveal the likely structural origins of the observed differences. Macroion mobility measurements were shown to be a useful and informative complement to functional studies in understanding complex macromolecular interactions. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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15. Covalent and Noncovalent Modifications Induce Allosteric Binding Behavior in a Monoclonal Antibody.
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Blake II, Robert C., Xia Li, Haini Yu, and Blake, Diane A.
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MONOCLONAL antibodies , *ALLOSTERIC regulation , *PROTEOLYSIS , *IMMUNOGLOBULINS , *BIOCHEMISTRY - Abstract
Detailed equilibrium binding studies were conducted on a monoclonal antibody (8A11) directed against UO22+ complexed with 2,9-dicarboxy-1,10-phenanthroline (DCP-UO22+). Covalent modification of 8A11 with amine-reactive derivatives of Cy5 or Alexa 488 altered the binding curves obtained with DCP-UO22+ from hyperbolic to sigmoidal, the latter characterized by Hill coefficients of 1.5-1.6. Binding curves obtained with DCP-UO22+ and the bivalent (Fab)2 or the monovalent Fab fragments derived from limited proteolysis of the covalently modified 8A11 were characterized by Hill coefficients of 1.2 and 1.0, respectively. Incubation of 8A11 with saturating concentrations of the Fab fragments of goat antibodies directed against the Fc portion of mouse IgG increased the affinity of the native 8A11 for DCP-UO22+ by 3-fold. Conversely, incubation of the 8A11 -Cy5 covalent conjugate with saturating concentrations of protein G (which likewise binds to the constant regions of mouse IgG) decreased the affinity of the primary antibody for DCP-UO22+ by 4-fold. In addition, the binding curves obtained with 8A11-Cy5 and DCP-UO22+ species changed from sigmoidal to hyperbolic at high concentrations of protein G. The presence of the antigen had a reciprocal effect on the binding of protein G to the 8A11-Cy5 conjugate; incubation of the 8A11-Cy5 conjugate with saturating concentrations of DCP-UO22+ decreased the affinity of the conjugate for protein G by 20-fold. These complex binding data were interpreted in terms of a free energy binding model in which (i) 2 mol of DCP-UO22+ and 1 mol of protein G bind to each mole of the 8A11-Cy5 conjugate, (ii) binding of the first equivalent of DCP-UO22+ to the antibody promotes the binding of the second equivalent of antigen in the absence of protein G, and (iii) DCP-UO22+ and protein G oppose each other's binding to the antibody. This is the first detailed description of the energetic balance of reciprocal binding events among the antigen binding sites and distant points on the constant portion of an immunoglobulin. [ABSTRACT FROM AUTHOR]
- Published
- 2007
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16. Quantifying Pseudomonas aeruginosa Adhesion to Contact Lenses1.
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Dang, Yen N.T., Rao, Aravinda, Kastl, Peter R., Blake II, Robert C., Schurr, Michael J., and Blake, Diane A.
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- 2003
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17. Allosteric Binding Properties of a Monoclonal Antibody and Its Fab Fragment.
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Blake II., Robert C., Delehanty, James B., Khosraviani, Mehraban, Yu, Haini, Jones, R. Mark, and Blake, Diane A.
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MONOCLONAL antibodies , *ALLOSTERIC regulation , *MICROBIAL proteins - Abstract
Detailed equilibrium binding studies were conducted on a monoclonal antibody directed against Pb(II) complexed with a protein conjugate of diethylenetriaminepentaacetic acid (DTPA). Binding curves obtained with DTPA and a cyclohexyl derivative of DTPA in the presence and absence of metal ions were consistent with the anticipated one-site homogeneous binding model. Binding curves obtained with aminobenzyl-DTPA or its complexes with Ca(II), Sr(II), and Ba(II) were highly sigmoidal, characterized by Hill coefficients of 2.3-6.5. Binding curves obtained with the Pb(II) and In(III) complexes of aminobenzyl-DTPA were hyperbolic, but in each case the apparent affinity of the antibody for the chelator-metal complex was higher in the presence of excess chelator than it was in the presence of excess metal ion. In the presence of excess chelator, the equilibrium dissociation constant for the binding of aminobenzyl-DTPA-Pb(II) to the antibody was 9.5 × 10[sup 10] M. Binding curves obtained with the Hg(II) and Cd(II) complexes of aminobenzyl-DTPA were biphasic, indicative of negative cooperativity. Further binding studies demonstrated that aminobenzyl-DTPA-Hg(II) opposed the binding of additional chelator-metal complexes to the antibody more strongly than did aminobenzyl-DTPA-Cd(II). The Fab fragment differed from the intact antibody only in that the apparent affinity of the Fab was generally lower for a given chelator-metal complex. These data are interpreted in terms of a model in which (i) aminobenzyl-DTPA and its complexes bind both to the antigen binding site and to multiple charged sites on the surface of the compact immunoglobulin; and (ii) the bound, highly charged ligands interact in a complicated fashion through the apolar core of the folded antibody. [ABSTRACT FROM AUTHOR]
- Published
- 2003
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18. Near real-time biosensor-based detection of 2,4-dinitrophenol
- Author
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Carter, Robert M., Blake II, Robert C., Nguyen, Trong D., and Bostanian, Levon A.
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BIOSENSORS , *DINITROPHENOL , *IMMUNOASSAY - Abstract
A fluorescent biosensor assay has been developed for near real-time detection of 2,4-dinitrophenol (DNP). The assay was based on fluorescent detection principles that allow for the analysis of antibody/antigen interactions in solution using the KinExA™ immunoassay instrument. Our KinExA™ consisted of a capillary flow observation cell containing a microporous screen that maintains a compact capture antigen-coated bead bed. The bead bed was comprised of polymethylmethacrylate (PMMA) beads coated with dinitrophenol–human serum albumin (DNP–HSA) conjugate. Phosphate buffered saline (PBS) solutions, containing various concentrations of free DNP, were incubated for 30 min with mouse anti-DNP monoclonal antibody to equilibrium. Solutions containing the DNP–monoclonal antibody complex and possible excess free antibodies were then passed over DNP–HSA labeled beads. The free monoclonal anti-DNP antibody, if available, was then bound to the DNP–HSA fixed on the beads. The system was then flushed with excess PBS to remove unbound reactants in the bead bed. The beads were then subjected to brief contact with PBS solutions containing goat anti-mouse fluorescein isothiocyanate (FITC)-labeled secondary antibody, once again, followed by a short PBS flush. The fluorescence was recorded during the addition of the FITC labeled secondary antibody to the bead bed through the final PBS flushing with the KinExA™. The amount of DNP detected could then be determined from the fluorescent slopes that were generated or by the remaining fluorescence that was retained on the beads after final PBS flushing of the system. This assay has been able to detect a minimum of 5 ng/ml of DNP in solution and can be adapted for other analytes of interest simply by changing the capture antigen and antibody pairs. [Copyright &y& Elsevier]
- Published
- 2003
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19. Respiratory enzymes of Thiobacillus ferrooxidans. Kinetic properties of an acid-stable iron...
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Blake, II, Robert C. and Shute, Elizabeth A.
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THIOBACILLUS ferrooxidans , *OXIDOREDUCTASES , *ENZYME kinetics - Abstract
Describes the discovery, partial purification, and relevant kinetic properties of an acid stable iron: rusticyanin oxidoreductase from cell-free extracts of T. ferrooxidans ATCC 23270. Description of experimental procedures; Results of experiment; Discussion of results; References.
- Published
- 1994
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20. Sulfolobus acidocaldarius Microvesicles Exhibit Unusually Tight Packing Properties as Revealed by Optical Spectroscopy.
- Author
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Bonanno, Alexander, Blake II, Robert C., and Chong, Parkson Lee-Gau
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OPTICAL spectroscopy , *MEMBRANE lipids , *POLAR molecules , *AMINO acid residues , *EXTRACELLULAR matrix proteins , *ETHER lipids - Abstract
In this study, we used optical spectroscopy to characterize the physical properties of microvesicles released from the thermoacidophilic archaeon Sulfolobus acidocaldarius (Sa-MVs). The most abundant proteins in Sa-MVs are the S-layer proteins, which self-assemble on the vesicle surface forming an array of crystalline structures. Lipids in Sa-MVs are exclusively bipolar tetraethers. We found that when excited at 275 nm, intrinsic protein fluorescence of Sa-MVs at 23 °C has an emission maximum at 303 nm (or 296 nm measured at 75 °C), which is unusually low for protein samples containing multiple tryptophans and tyrosines. In the presence of 10–11 mM of the surfactant n-tetradecyl-β-d-maltoside (TDM), Sa-MVs were disintegrated, the emission maximum of intrinsic protein fluorescence was shifted to 312 nm, and the excitation maximum was changed from 288 nm to 280.5 nm, in conjunction with a significant decrease (>2 times) in excitation band sharpness. These data suggest that most of the fluorescent amino acid residues in native Sa-MVs are in a tightly packed protein matrix and that the S-layer proteins may form J-aggregates. The membranes in Sa-MVs, as well as those of unilamellar vesicles (LUVs) made of the polar lipid fraction E (PLFE) tetraether lipids isolated from S. acidocaldarius (LUVPLFE), LUVs reconstituted from the tetraether lipids extracted from Sa-MVs (LUVMV) and LUVs made of the diester lipids, were investigated using the probe 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan). The generalized polarization (GP) values of Laurdan in tightly packed Sa-MVs, LUVMV, and LUVPLFE were found to be much lower than those obtained from less tightly packed DPPC gel state, which echoes the previous finding that the GP values from tetraether lipid membranes cannot be directly compared with the GP values from diester lipid membranes, due to differences in probe disposition. Laurdan's GP and red-edge excitation shift (REES) values in Sa-MVs and LUVMV decrease with increasing temperature monotonically with no sign for lipid phase transition. Laurdan's REES values are high (9.3–18.9 nm) in the tetraether lipid membrane systems (i.e., Sa-MVs, LUVMV and LUVPLFE) and low (0.4–5.0 nm) in diester liposomes. The high REES and low GP values suggest that Laurdan in tetraether lipid membranes, especially in the membrane of Sa-MVs, is in a very motionally restricted environment, bound water molecules and the polar moieties in the tetraether lipid headgroups strongly interact with Laurdan's excited state dipole moment, and "solvent" reorientation around Laurdan's chromophore in tetraether lipid membranes occurs very slowly compared to Laurdan's lifetime. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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21. Expression and purification of the HTLV-1 transforming protein Tax.
- Author
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Evans, Whitney, Carriere, Patrick, Weber, Morgan, Preyan, Lynez, Korona, Boguslawa, Blake,II., Robert C., Traina-Dorge, Vicki, and Shuh, Maureen
- Subjects
HTLV ,PROTEIN synthesis - Abstract
An abstract of the research paper titled,"Expression and purification of the of the Human T-cell leukemia virus type 1 (HTLV-1) transforming protein Tax ," is presented.
- Published
- 2011
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22. The Multicenter Aerobic Iron Respiratory Chain of Acidithiobacillus ferrooxidans Functions as an Ensemble with a Single Macroscopic Rate Constant.
- Author
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Ting-Feng Li, Painter, Richard G., Ban, Bhupal, and Blake II, Robert C.
- Subjects
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THIOBACILLUS ferrooxidans , *CHARGE exchange , *CYTOCHROMES , *REDUCTION potential , *OXIDATION-reduction potential - Abstract
Electron transfer reactions among three prominent colored proteins in intact cells of Acidithiobacillus ferrooxidans were monitored using an integrating cavity absorption meter that permitted the acquisition of accurate absorbance data in suspensions of cells that scattered light. The concentrations of proteins in the periplasmic space were estimated to be 350 and 25 mg/ml for rusticyanin and cytochrome c, respectively; cytochrome a was present as one molecule for every 91 nm² in the cytoplasmic membrane. All three proteins were rapidly reduced to the same relative extent when suspensions of live bacteria were mixed with different concentrations of ferrous ions at pH 1.5. The subsequent molecular oxygen-dependent oxidation of the multicenter respiratory chain occurred with a single macroscopic rate constant, regardless of the proteins' in vitro redox potentials or their putative positions in the aerobic iron respiratory chain. The crowded electron transport proteins in the periplasm of the organism constituted an electron conductive medium where the network of protein interactions functioned in a concerted fashion as a single ensemble with a standard reduction potential of 650 mV. The appearance of product ferric ions was correlated with the reduction levels of the periplasmic electron transfer proteins; the limiting first-order catalytic rate constant for aerobic respiration on iron was 7,400 s-1. The ability to conduct direct spectrophotometric studies under noninvasive physiological conditions represents a new and powerful approach to examine the extent and rates of biological events in situ without disrupting the complexity of the live cellular environment. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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23. Kinetic rate constant for electron transfer between ferrous ions and novel Rusticyanin isoform in Acidithiobacillus ferrooxidans
- Author
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Ida, Chigusa, Sasaki, Kazuhiro, Ando, Kikazu, Blake II, Robert C., Saiki, Hiroshi, and Ohmura, Naoya
- Subjects
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CHARGE exchange , *IRON ions , *METAL ions , *ELECTRONS , *PHYSICAL & theoretical chemistry - Abstract
Here we report the kinetic rate constant for electron transfer from ferrous ions to a novel rusticyanin isoform in Acidithiobacillus ferrooxidans. The second order rate constant for this isoform is shown to be approximately one half that of the previously known type, 0.09 M−1s−1 vs. 0.14 M−1s−1. [Copyright &y& Elsevier]
- Published
- 2003
- Full Text
- View/download PDF
24. Novel monoclonal antibodies with specificity for chelated uranium(VI): isolation and binding properties.
- Author
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Blake II RC, Pavlov AR, Khosraviani M, Ensley HE, Kiefer GE, Yu H, Li X, and Blake DA
- Subjects
- Animals, Antibodies, Monoclonal isolation & purification, Cattle, Dose-Response Relationship, Drug, Female, Mice, Mice, Inbred BALB C, Protein Binding, Serum Albumin, Bovine metabolism, Antibodies, Monoclonal metabolism, Antibody Specificity, Binding Sites, Antibody, Chelating Agents metabolism, Uranium immunology, Uranium metabolism
- Abstract
A derivative of 1,10-phenanthroline that binds to UO(2)(2+) with nanomolar affinity was found to be a very effective immunogen for the generation of antibodies directed toward chelated complexes of hexavalent uranium. This study describes the synthesis of 5-isothiocyanato-1,10-phenanthroline-2,9-dicarboxylic acid and its use in the generation and functional characterization of a group of monoclonal antibodies that recognize the most soluble and toxic form of uranium, the hexavalent uranyl ion (UO(2)(2+)). Three different monoclonal antibodies (8A11, 10A3, and 12F6) that recognize the 1:1 complex between UO(2)(2+) and 2,9-dicarboxy-1,10-phenanthroline (DCP) were produced by the injection of BALB/c mice with DCP-UO(2)(2+) covalently coupled to a carrier protein. Equilibrium dissociation constants for the binding of DCP-UO(2)(2+) to antibodies 8A11, 10A3, and 12F6 were 5.5, 2.4, and 0.9 nM, respectively. All three antibodies bound the metal-free DCP with roughly 1000-fold lower affinity. The second-order rate constants for the bimolecular association of each antibody with soluble DCP-UO(2)(2+) were in the range of 1 to 2 x 10(7) M(-1) s(-1). Binding studies conducted with structurally related chelators and 21 metal ions demonstrated that each of these three antibodies was highly specific for the soluble DCP-UO(2)(2+) complex. Detailed equilibrium binding studies conducted with three other derivatives of DCP, either complexed with UO(2)(2+) or metal-free, suggested that the antigen binding sites on the three antibodies have significant functional and structural similarities. Biomolecules that bind specifically to uranium will be at the heart of any new biotechnology developed to monitor and control uranium contamination. The three antibodies described herein possess sufficient affinity and specificity to support the development of immunoassays for hexavalent uranium in environmental and clinical samples.
- Published
- 2004
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