Robert C. Blake

METAL BINDING PROPERTIES OF A MONOCLONAL ANTIBODY DIRECTED TOWARD METAL-CHELATE COMPLEXES

A monoclonal antibody that recognizes cadmium-EDTA complexes has been produced by the injection of BALB/c mice with a metal-chelate complex covalently coupled to a carrier protein. The ability of purified antibody to recognize 16 different metal-EDTA complexes was assessed by measuring equilibrium binding constants using a KinExA™ immunoassay instrument. The antibody bound to cadmium- and mercury-EDTA complexes with equilibrium dissociation constants of 21 and 26 nM, respectively. All other metal-EDTA complexes tested, including those of Mn(II), In(III), Ni(II), Zn(II), Co(II), Cu(II), Ag(I), Fe(III), Pb(II), Au(III), Tb(III), Ga(III), Mg(II), and Al(III) bound with affinities from 20- to 40,000-fold less than that determined for the cadmium-EDTA complex. With the exception of mercury and magnesium, the binding of divalent metal-chelate complexes was well-correlated with the size of the metal ion. The amino acid sequences of the heavy and light chain variable regions were deduced from polymerase chain reaction-amplified regions of the corresponding genes and subsequently used to construct molecular models of the antigen binding region. The key residue for cadmium binding in the model for 2A81G5 appeared to be histidine 96 in the heavy chain.

In situ Spectroscopy on Intact Leptospirillum ferrooxidans Reveals that Reduced Cytochrome 579 is an Obligatory Intermediate in the Aerobic Iron Respiratory Chain.

Electron transfer reactions among colored cytochromes in intact bacterial cells were monitored using an integrating cavity absorption meter that permitted the acquisition of accurate absorbance data in suspensions of cells that scatter light. The aerobic iron respiratory chain of Leptospirillum ferrooxidans was dominated by the redox status of an abundant cellular cytochrome that had an absorbance peak at 579u2009nm in the reduced state. Intracellular cytochrome 579 was reduced within the time that it took to mix a suspension of the bacteria with soluble ferrous iron at pH 1.7. Steady state turnover experiments were conducted where the initial concentrations of ferrous iron were less than or equal to that of the oxygen concentration. Under these conditions, the initial absorbance spectrum of the bacterium observed under air-oxidized conditions was always regenerated from that of the bacterium observed in the presence of Fe(II). The kinetics of aerobic respiration on soluble iron by intact L. ferrooxidans conformed to the Michaelis–Menten formalism, where the reduced intracellular cytochrome 579 represented the Michaelis complex whose subsequent oxidation appeared to be the rate-limiting step in the overall aerobic respiratory process. The velocity of formation of ferric iron at any time point was directly proportional to the concentration of the reduced cytochrome 579. Further, the integral over time of the concentration of the reduced cytochrome was directly proportional to the total concentration of ferrous iron in each reaction mixture. These kinetic data obtained using whole cells were consistent with the hypothesis that reduced cytochrome 579 is an obligatory steady state intermediate in the iron respiratory chain of this bacterium. The capability of conducting visible spectroscopy in suspensions of intact cells comprises a powerful post-reductionist means to study cellular respiration in situ under physiological conditions for the organism.

Near real-time biosensor-based detection of 2,4-dinitrophenol

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.

Does aporusticyanin mediate the adhesion of Thiobacillus ferrooxidans to pyrite

Abstract The adhesion of Thiobacillus ferrooxidans to pyrite was quantified by electrical impedance measurements. Cells grown on soluble iron adhered specifically and with high affinity to pyrite, exhibiting an equilibrium dissociation constant of 5×10 −15 M cells. Purposeful manipulation of individual cells using optical trapping techniques revealed that 92% of the iron-grown cells adhered to pyrite with a force greater than 5.2 pN, the maximum force exerted by the trap. In contrast, cells grown on sulfur adhered to pyrite with lower affinity, and 91% of sulfur-grown cells were dissociated from pyrite with an average force of 3.6 pN. Purified recombinant aporusticyanin and intact cells of T. ferrooxidans showed an identical pattern of adhesion to the same minerals. The addition of ferrous ions or organic chelators to the binding mixture prevented the binding of either aporusticyanin or intact cells to pyrite. Preincubation of either the pyrite alone or both the pyrite and the cells with exogenous aporusticyanin inhibited the adhesion of cells to pyrite by 41% and 60%, respectively. A His85Ala mutant apoprotein bound much less tightly to pyrite than did the wild type aporusticyanin. These observations are consistent with a model where aporusticyanin located on the surface of the bacterial cell acts as a mineral-specific receptor for the initial adhesion of T. ferrooxidans to pyrite. Binding of the apoprotein to solid pyrite is accomplished in part by coordination of the unoccupied copper ligands with an iron atom at the exposed edge of the pyrite crystal lattice.

A fluorescent biosensor for detection of zearalenone.

ABSTRACT A fluorescent biosensor was developed on a KinExATM flow spectrofluorimeter for the near real-time detection of soluble zeaalenone. Briefly, solutions of zearalenone and a monoclonal antibody directed against a protein conjugate of zearalenone, were incubated for thirty minutes to permit equilibrium binding to occur. The reaction mixture was then passed over a packed column of small beads (98 μm) whose surfaces were coated with a covalent conjugate of zearalenone and bovine serum albumin (BSA). Following a short wash with buffer to remove excess unbound primary reagents, the packed beads were subjected to a brief contact with fluorescein isothiocyanate-labeled polyclonal secondary antibody directed against the primary monoclonal, once again followed by a short wash. As this assay depends on the ability of soluble antigen to compete with immobilized antigen, increasing concentrations of zearalenone result in decreasing fluorescence observed on the bead pack. This assay is rapid (≅ 60 minutes) and can be adapted to various other analytes of interest.

Kinetic rate constant for electron transfer between ferrous ions and novel Rusticyanin isoform in Acidithiobacillus ferrooxidans.

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(-1)s(-1) vs. 0.14 M(-1)s(-1).

The Multicenter Aerobic Iron Respiratory Chain of Acidithiobacillus ferrooxidans Functions as an Ensemble with a Single Macroscopic Rate Constant

Background: The order of electron transfer in the respiratory chain of Acidithiobacillus ferrooxidans is unknown. Results: The multicenter respiratory chain functioned as an ensemble with a single macroscopic rate constant. Conclusion: The crowded respiratory proteins behaved as would a single protein with a common standard reduction potential of 650 mV. Significance: In situ spectroscopy constitutes a new and effective means to study cellular respiration. 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.

[27] Assays for cytochrome P-450 peroxygenase activity

Publisher Summary This chapter discusses assays for cytochrome P- 450 peroxygenase activity. Toluene, which yields benzyl alcohol in the cytochrome P- 450-catalyzed peroxygenase reaction, is a useful substrate in mechanistic studies. To determine benzyl alcohol formation at fixed time intervals, a gas chromatographic assay is employed. Cytochrome P- 450 is versatile enzyme that catalyzes numerous types of chemical reactions with a variety of substrates, including the hydroxylation of xenobiotics, such as drugs, pesticides, dyes, organic solvents, anesthetics, and carcinogens, as well as naturally occurring lipids, including steroids, fatty acids, and prostaglandins. Molecular oxygen serves as the natural donor when electrons are supplied to cytochrome P- 450 by nicotinamide adenine dinucleotide phosphate (NADPH) via proteins that serve as electron carriers. In the case of liver microsomes or the reconstituted enzyme system, NADPH-cytochrome- P- 450 reductase serves as the carrier and in the overall reaction equimolar amounts of substrate, O 2 , and NADPH are consumed and equimolar amounts of hydroxylated substrate, H 2 O, and NADP are formed. Molecular oxygen can be replaced by a variety of hydroperoxides and related artificial donors. Following the discovery of the ability of cytochrome P- 450-containing microsomal suspensions to promote the oxygenation of an organic substrate at the expense of an alkyl hydroperoxide, other oxidants, such as peroxy acids, periodate, iodosobenzene, iodobenzene diacetate, and N-oxides, are found to function in a similar capacity.

In situ spectroscopy reveals that microorganisms in different phyla use different electron transfer biomolecules to respire aerobically on soluble iron

Absorbance spectra were collected on 12 different live microorganisms, representing six phyla, as they respired aerobically on soluble iron at pH 1.5. A novel integrating cavity absorption meter was employed that permitted accurate absorbance measurements in turbid suspensions that scattered light. Illumination of each microorganism yielded a characteristic spectrum of electrochemically reduced colored prosthetic groups. A total of six different patterns of reduced-minus-oxidized difference spectra were observed. Three different spectra were obtained with members of the Gram-negative eubacteria. Acidithiobacillus, representing Proteobacteria, yielded a spectrum in which cytochromes a and c and a blue copper protein were all prominent. Acidihalobacter, also representing the Proteobacteria, yielded a spectrum in which both cytochrome b and a long-wavelength cytochrome a were clearly visible. Two species of Leptospirillum, representing the Nitrospirae, both yielded spectra that were dominated by a cytochrome with a reduced peak at 579 nm. Sulfobacillus and Alicyclobacillus, representing the Gram-positive Firmicutes, both yielded spectra dominated by a-type cytochromes. Acidimicrobium and Ferrimicrobium, representing the Gram-positive Actinobacteria, also yielded spectra dominated by a-type cytochromes. Acidiplasma and Ferroplasma, representing the Euryarchaeota, both yielded spectra dominated by a ba3-type of cytochrome. Metallosphaera and Sulfolobus, representing the Crenarchaeota, both yielded spectra dominated by the same novel cytochrome as that observed in the Nitrospirae and a new, heretofore unrecognized redox-active prosthetic group with a reduced peak at around 485 nm. These observations are consistent with the hypothesis that individual acidophilic microorganisms that respire aerobically on iron utilize one of at least six different types of electron transfer pathways that are characterized by different redox-active prosthetic groups. In situ absorbance spectroscopy is shown to be a useful complement to existing means of investigating the details of energy conservation in intact microorganisms under physiological conditions.

Re-Engineering Antibodies for Optimum Performance in Uranium Sensors

Our laboratory has developed antibody-based sensors that provide on-site, near real-time information about uranium in environmental samples. The monoclonal antibody at the heart of this technology (clone 12F6) specifically recognizes U(VI) complexed to a chelator (2,-9-dicarboxyl-1-10-phenanthroline). Mutagenesis of a single amino acid in the 12F6 antibody light chain significantly increased its binding to chelated U(VI). Insertion of a gold-binding peptide sequence enhanced the antibody’s ability to complex with the gold nanoparticles, a process required for the development of simple lateral flow strips for uranium monitoring.