ABSTRACT
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.
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.
