PubMed · 14101980
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M D MELAMED, N M GREEN. 1963. AVIDIN. 2. PURIFICATION AND COMPOSITION.. https://doi.org/10.1042/bj0890591
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Conjugates of avidin with ferrocene and with microperoxidase 8 have been used as electrochemically active molecular building blocks. Assemblies of the conjugates with biotinylated glucose oxidase or lactate oxidase on gold electrodes were tested as enzyme sensors for glucose and lactate. The electrochemical detection is based either on ferrocene-mediated oxidation of the substrate in oxygen-free solution, or on microperoxidase-catalysed reduction of H2O2 which is enzymatically produced from the substrate and molecular oxygen. Glucose and lactate were detectable with both detection principles in concentrations down to 1 or 0.1 mM, respectively. The molecular architecture concept allows quick adaptation of the sensors to other analytes, and it provides a platform for arrays of sensors with different selectivity.
Single-molecule measurements of the activities of a variety of enzymes show that rates of catalysis may vary markedly between different molecules in putatively homogeneous enzyme preparations. We measured the rate at which purified Escherichia coli RNA polymerase moves along a approximately 2650-bp DNA during transcript elongation in vitro at 0.5 mm nucleoside triphosphates. Individual molecules of a specifically biotinated RNA polymerase derivative were tagged with 199-nm diameter avidin-coated polystyrene beads; enzyme movement along a surface-linked DNA molecule was monitored by observing changes in bead Brownian motion by light microscopy. The DNA was derived from a naturally occurring transcription unit and was selected for the absence of regulatory sequences that induce lengthy pausing or termination of transcription. With rare exceptions, individual enzyme molecules moved at a constant velocity throughout the transcription reaction; the distribution of velocities across a population of 140 molecules was unimodal and was well fit by a Gaussian. However, the width of the Gaussian, sigma = 6.7 bp/s, was considerably larger than the precision of the velocity measurement (1 bp/s). The observations show that different transcription complexes have differences in catalytic rate (and thus differences in structure) that persist for thousands of catalytic turnovers. These differences may provide a parsimonious explanation for the complex transcription kinetics observed in bulk solution.
The objective of this work was to incorporate cytoadhesive properties into poly(methylmethacrylate) (PMMA) for potential applications in highly localized tissue-specific drug delivery. First, the PMMA was chemically modified by aminolysis to yield amine-terminated surfaces. X-ray photoelectron microscopy confirmed the presence of surface nitrogen entities, and the distribution of amine groups was found to be relatively uniform, as characterized by atomic force microscopy. The availability of these groups for attachment of biologically active molecules was characterized by fluorescence microscopy after immobilization of avidin-FITC. To render the PMMA cytoadhesive, avidin molecules were conjugated to the amine-terminated surfaces with a hydroxy-succinimide-catalyzed carbodiimide reagent and biotin-labeled lectins (tomato, which binds selectively to Caco-2 cells, and peanut, an unrelated lectin) subsequently were attached utilizing avidin-biotin chemistry. Cytoadhesive activity was evaluated by characterizing the interactions between microfabricated PMMA particles and Caco-2 monolayers. After 15-, 30-, 60-, and 120-min incubation periods, the tomato lectin-conjugated PMMA showed a two to sixfold increase in Caco-2 cell recognition over control particles. Furthermore, the stability of the cytoadhesive PMMA interactions appeared to be three to seven times greater than that of the control surfaces. These findings demonstrate that cytoadhesive properties of modified PMMA, making this novel bioactive polymer very promising for applications in targeted drug delivery.