Michelle J Cannon

University of Utah, Salt Lake City, Utah, United States

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Publications (9)22.61 Total impact

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    ABSTRACT: Surface plasmon resonance imaging systems, such as Flexchip from Biacore, are capable of monitoring hundreds of reaction spots simultaneously within a single flow cell. Interpreting the binding kinetics in a large-format flow cell presents a number of potential challenges, including accounting for mass transport effects and spot-to-spot sample depletion. We employed a combination of computer simulations and experimentation to characterize these effects across the spotted array and established that a simple two-compartment model may be used to accurately extract intrinsic rate constants from the array under mass transport-limited conditions. Using antibody systems, we demonstrate that the spot-to-spot variability in the binding kinetics was <9%. We also illustrate the advantage of globally fitting binding data from multiple spots within an array for a system that is mass transport limited.
    No preview · Article · Feb 2008 · Analytical Biochemistry
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    ABSTRACT: To gauge the experimental variability associated with Biacore analysis, 36 different investigators analyzed a small molecule/enzyme interaction under similar conditions. Acetazolamide (222 g/mol) binding to carbonic anhydrase II (CAII; 30000 Da) was chosen as a model system. Both reagents were stable and their interaction posed a challenge to measure because of the low molecular weight of the analyte and the fast association rate constant. Each investigator created three different density surfaces of CAII and analyzed an identical dilution series of acetazolamide (ranging from 4.1 to 1000 nM). The greatest variability in the results was observed during the enzyme immobilization step since each investigator provided their own surface activating reagents. Variability in the quality of the acetazolamide binding responses was likely a product of how well the investigators' instruments had been maintained. To determine the reaction kinetics, the responses from the different density surfaces were fit globally to a 1:1 interaction model that included a term for mass transport. The averaged association and dissociation rate constants were 3.1+/-1.6 x 10(6)M(-1)s(-1) and 6.7+/-2.5 x 10(-2)s(-1), respectively, which corresponded to an average equilibrium dissociation constant (K(D) of 2.6+/-1.4 x 10(-8)M. The results provide a benchmark of variability in interpreting binding constants from the biosensor and highlight keys areas that should be considered when analyzing small molecule interactions.
    Full-text · Article · Aug 2004 · Analytical Biochemistry
  • Michelle J Cannon · Angela D Williams · Ronald Wetzel · David G Myszka
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    ABSTRACT: We used surface plasmon resonance biosensors to evaluate the kinetics associated with the initial events of beta-amyloid (Abeta) fibril elongation. Fibrils were immobilized on the sensor chip surface and extended by exposure to soluble Abeta(1-40) peptide. The fibril surfaces bound Congo red, a marker for beta sheet structures, and exhibited a slow linear background decay that is consistent with fibril depolymerization. Sonicated fibrils supported elongation better than unsonicated fibrils, which is consistent with fibril extension reactions. The kinetic data revealed that peptide association and dissociation occurred in multiple steps. Kinetic rate constants for fibril extension were determined by globally fitting the response data with a three-step polymerization model. In the first step, the soluble peptide binds to the growing fibril tip in a readily reversible reaction. The subsequent steps likely allow bound peptide to be stabilized into the growing fiber through postbinding transitional events. Using a mutant peptide, F19P Abeta(1-40), we illustrate how the biosensor assay can be used to probe structure/function relationships of fibril elongation.
    No preview · Article · Jun 2004 · Analytical Biochemistry
  • James M McGreevy · Michelle J Cannon · Charles B Grissom
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    ABSTRACT: We examined the usefulness of a new agent in the mapping and dissection of inguinal lymph nodes in the pig. Cy5-cobalamin bioconjugate is blue under visible light and fluoresces brilliant red with laser stimulation. The wavelength of the emitted red light is sufficiently long that it is visible through blood, subcutaneous fat, and fascia. Currently available surgical techniques of minimally invasive dissection are well suited for using fluorescent detection in a dark operating field with minimal modification of an existing Hopkins surgical telescope. We tested this concept in the live post-adolescent, female, nonlactating pig (30 kg). We insufflated the subcutaneous tissue over the groin and inserted three ports (1 x 10 mm and 2 x 5 mm) for dissection. We injected the Cy5-cobalamin bioconjugate in a dermal location on the hind limb. A HeNe laser stimulated the CobalaFluor in the lymphatics and the emitted fluorescence passed through a holographic notch filter to a three-chip camera. Under white light, the lymphatic trunks and the sentinel node were visualized within minutes of injection. Both the lymphatic trunks and the node fluoresced bright red under stimulation with red laser light. These preliminary studies establish the potential usefulness of this new agent in lymphatic mapping. This novel technology might be useful in visualizing cancers that spread to regional lymph nodes. This technique has the potential to map the lymphatic drainage and to identify the presence of malignant cells in that drainage with currently available minimally invasive technology.
    No preview · Article · Jun 2003 · Journal of Surgical Research
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    ABSTRACT: Surface plasmon resonance biosensor analysis was used to evaluate the thermodynamics and binding kinetics of naturally occurring and synthetic cobalamins interacting with vitamin B(12) binding proteins. Cyanocobalamin-b-(5-aminopentylamide) was immobilized on a biosensor chip surface to determine the affinity of different cobalamins for transcobalamin, intrinsic factor, and nonintrinsic factor. A solution competition binding assay, in which a surface immobilized cobalamin analog competes with analyte cobalamin for B(12) protein binding, shows that only recombinant human transcobalamin is sensitive to modification of the corrin ring b-propionamide of cyanocobalamin. A direct binding assay, where recombinant human transcobalamin is conjugated to a biosensor chip, allows kinetic analysis of cobalamin binding. Response data for cyanocobalamin binding to the transcobalamin protein surface were globally fitted to a bimolecular interaction model that includes a term for mass transport. This model yields association and dissociation rate constants of k(a) = 3 x 10(7) M(-1) s(-1) and k(d) = 6 x 10(-4) s(-1), respectively, with an overall dissociation constant of K(D) = 20 pM at 30 degrees C. Transcobalamin binds cyanocobalamin-b-(5-aminopentylamide) with association and dissociation rates that are twofold slower and threefold faster, respectively, than transcobalamin binding to cyanocobalamin. The affinities determined for protein-ligand interaction, using the solution competition and direct binding assays, are comparable, demonstrating that surface plasmon resonance provides a versatile way to study the molecular recognition properties of vitamin B(12) binding proteins.
    No preview · Article · Jul 2002 · Analytical Biochemistry

  • No preview · Article · Jan 2002 · Analytical Biochemistry
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    ABSTRACT: Fluorescent derivatives of cobalamin have been prepared by linking fluorophores to cobalamin through a propylamide spacer. Fluorescein, naphthofluorescein, and Oregon Green derivatives have been prepared in good yield by reaction of the fluorophore NHS-ester with beta-(3-aminopropyl)cobalamin to form fluorescent cobalamin conjugates (CobalaFluors) that are potentially suitable for the in vitro and in vivo imaging of transcobalamin receptors on cancer cells.
    Full-text · Article · Apr 2001 · Organic Letters
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    ABSTRACT: Fluorescent analogs of cobalamin (vitamin B12) have been developed as diagnostic markers of cancer cells. These compounds are recognized by transcobalamin, a cobalamin transport protein, with high affinity, as shown by surface plasmon resonance. The cellular sequestration and gross distribution of fluorescent cobalamin bioconjugates in breast tissue is being examined by epifluorescence microscopy. The distribution of each compound is being evaluated in proliferative and non-proliferative tissue, i.e. normal tissue and breast carcinoma. The results of preliminary studies suggest that fluorescent analogs of cobalamin may be a useful tool in therapeutic breast operations to define tumor margins and to distinguish neoplastic breast tissue from healthy breast tissue.
    No preview · Article · May 2000 · Proceedings of SPIE - The International Society for Optical Engineering
  • Silvia L. Porello · Michelle J. Cannon · Sheila S. David
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    ABSTRACT: The Escherichia coli DNA repair enzyme MutY plays an important role in the recognition and repair of 7, 8-dihydro-8-oxo-2'-deoxyguanosine: 2'-deoxyadenosine (OG:A) mismatches in DNA [Michaels et al. (1992) Proc. Natl. Acad. Sci. U.S. A. 89, 7022-7025]. MutY prevents mutations due to misincorporation of A opposite OG during DNA replication by removing the adenine base. This enzyme has significant sequence homology with the [4Fe-4S]2+ cluster-containing DNA repair enzyme, endonuclease III [Michaels et al. (1990) Nucleic AcidsRes. 18, 3841-3845]. In the present study, we have investigated the importance of cluster assembly in folding of MutY. MutY was denatured and then refolded in the presence or absence of ferrous and sulfide ions. Denatured MutY can refold in the presence of ferrous and sulfide ions to provide active enzyme. This suggests the cluster can self-assemble and that this process is facile in vitro. Interestingly, CD spectra and Tm measurements of MutY refolded with and without ferrous and sulfide ions are essentially identical, implying that assembly of the cluster is not required for MutY folding. Additionally, Tm measurements indicated that the [4Fe-4S]2+ cluster does not contribute significantly to the overall thermal stability of MutY. Refolded forms of MutY which lack the cluster are unable to perform the adenine glycosylase function and bind to DNA. However, these inactive folded forms regain activity by addition of ferrous and sulfide ions. This indicates that the Fe-S cluster may have a superficial location, allowing for its assembly after folding. More importantly, these results provide evidence that the presence of the [4Fe-4S]2+ cluster is critical for the specific recognition of substrate DNA necessary for the adenine glycosylase activity of MutY.
    No preview · Article · May 1998 · Biochemistry