Fluorophores

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Fluorophores

• Chapter 3

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Intrinsic Fluorophores

• Aromatic amino acids tryptophan, tyrosine,
  phenylalanine
• Tryptophan indole group dominates absorbance
  and fluorescence
• Tyrosine - emission often quenched
• Typrophan fluorescence
• sensitive to local environment
• Can be quenched by NH3, COOH, and protonated
  histidine residues

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Fluorescent Enzyme Cofactors

• NADH highly fluorescent, ex 340 nm, em 460
  nm
• Nicotinamide group is quenched by adenine
• Binding of NADH to proteins enhances QY, 4-fold
• Oxidized NADH (NAD) non-fluorescent
• Pyridoxal Phosphate sensitive to protein
  binding, pH, participation in enzyme reaction
• FAD fluorescent, but reduced form (FADH)
  non-fluorescent
• Yt base of Yeast tRNA
• Autofluoresence from tissues collagen and
  elastin. Excite 340 nm and emitt near 405. HP
  cross-links in collagen may play a role.

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Extrinsic Fluorophores

• Labeling proteins or biological macromolecule
  with an extrinsic probe
• Longer wavelengths desirable reduce background
  fluorescence from naturally fluorescent groups
• Molecular Probes Catalog large number of probes
  for many different assays

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Protein Labeling Reagents

• Covalent probes couple to amine, sulfhydryl, or
  histidine side chains
• Dansyl Chloride (DNS-Cl) favorable lifetime (10
  ns). Acceptor for FRET from intrinsic probes
  (Absorb near 350 nm) tryptophan. Probe sensitive
  to solvent (emission near 520 nm)
• Fluoresceins and rhodamines long wavelength
  absorption (480 and 560 nm)
• High absorption E 80,000 M-1 cm-1
• Wide variety of reactive derivatives
  Iodoacetamides and maleimides sulfhydryl groups
• N-hydroxysuccinimide, sulfonyl chloride -
  labeling amides
• Can be mixed or single isomers
• Microscopy and immunoassays linkages to
  antibodies
• Lifetime near 4 ns and not sensitive to solvent
  polarity
• Association of small labeled molecules with
  proteins via polarization

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Role of Stokes Shift in Protein Labeling

• Fluorescein tendency to self quench, because
  of small Stokes shift susceptible to FRET if
  multiple probes attached to a single protein
• DNS-Cl higher stokes shift, but hydrophobic
  which can result in protein aggregation
• BODIPY boron containing fluorophore, high
  quantum yield and insensitivity to solvent
  polarity and pH. Disadvantage small stokes
  shift
• Solvent sensitive prodan, spectral shift
  observed during membrane phase-transition

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Noncovalent Protein-Labeling

• ANS and TNS weakly fluorescent in water and
  fluorescence enhancement when bound to proteins
• FlAsH fluoresceine biarsenical hairpin binding

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1-anilinonaphthalene-8-sulfonic acid
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Membrane Probes

• DPH (1,6-diphenyl-1,3,5-hexatriene) all
  fluorescence due to DPH in membrane environment
• Lipid probes attached to fatty acid side chains
  or phospholipids
• Pyene excimer formation determine diffusive
  processes in membrane
• Attachment of fluorescein or rhodamine to long
  lipid side chain (Texas Red- PE)
• Membrane potential probes partitioning of the
  dye from the water to the membrane phase,
  reorientation of the dyes from the membrane,
  aggregation of the dyes, insensitivity of the
  dyes to the electric field

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Red and Near-Infrared (NIR) Dyes

• Cyanine dyes decreased background over
  autoflourescence
• Excitation with simple laser sources solid
  state laser diodes
• Cy-3, Cy-5, Cy-7 absorption and emission near
  650 nm, small stokess shift (30 nm)
• Rhodamine 800, IR-125, Thiazole Orange (ex, 735
  nm, binds DNA)

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DNA Probes

• Weakly fluorescent in the UV
• Many dyes that bind ethidium bromide, 30-fold
  enhancement upon binding, lifetime increases 1.7
  to 20 ns
• EtBr interacts between the base-pairs of the
  double-helical DNA
• DAPI minor groove binding
• Ethidium homodimer and TOTO-1 higher affinity
  binding

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Chemical Sensing Probes

• Detection of ions (Cl-, Na, Ca)
• Ratiometric probes Fura 2, Ca can be
  calculated by spectra shift
• Calcium green enhancement but no spectral shift
• Fluorescence microscopy trap in cell with
  hydrolysis of cell permeable esters or
  microinjection

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Special Probes

• Non-fluoroscent until chemical change can
  follow enzyme reaction which induces chemical
  change. 7-UmP activity of alkaline phosphatase
  ELISA assay
• B-galactosidase activity marker of gene
  expression in cells 7-hydoxycoumarin

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• Enhancement upon reactivity with amines protein
  sequencing, concentration, detection

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• Follow enzymatic cleavage reaction by FRET,
  Phosopholipid hydrolysis by lipases
• Fluorogenic precipitate probe phosphatase or
  galactosidase, hydrolysis ppt the fluorogenic
  group becomes fluorescent

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Structural Analogs of Biomolecules

• Cholesterol dehydroergosterol can be used to
  study cholesterol interactions in the membrane
• Estradiol azetrahydrochrysene, analysis of
  estrogen receptors in cancer cells
• ATP analogs ?ATP and mantATP, Formycin
  (adenosine)

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Viscosity Probes

• Probes that distort in the excited state to form
  intermolecular charge-transfer states
• Level of viscosity can be determined by
  fluorescence intensity

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Fluorescent Proteins

• Phycobiliproteins (blue-green and red algae)
  large proteins made up of a number of subunits
• High density of chromophores high extinction
  coefficient and high fluorescence (30X
  fluoroscein)
• Water soluable and stable
• Long wavelength emission
• Good Stokes shift
• Sensitive to illumination excitation of more
  than one chromophore per protein

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Phytofluors

• Light sensitive proteins present in
  photosynthetic organisms
• Non-fluorescent fluorophore converts between
  two stable forms
• Phycoerythrobilin pigment needs to be added -
  which needs to be transported into cells

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Green Fluorescent Protein

• Jellyfish (Aequorea victoria) highly
  fluorescent group within a constrained protected
  region of the protein (B-Barrel)
• Synthesize GFP fusion proteins in cells,
  organisms
• Many different mutants designed with different
  fluorescence spectral properties (BFP, YFP, CFP)

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Long-lifetime Probes

• Pyrene 400 ns in degassed organic solvent,
  labeled macromolecules displays multiexponential
  decays, photochemical changes
• Coronene conjugated to lipids

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Lanthanides

• Emission from transitions involving 4f orbitals,
  forbidden transitions
• Low absorption coefficient
• Long lifetime
• Excited through chelated organic ligands
• Can subsitute for Ca in Ca dependent proteins
• Can coupled to proteins by introducing a site
  which chelates lanthanides

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Lanthanides Immunoassays

• Lanthanides continue to emitt fluorescence
  following the autofluorescence decay
• Intensity measured over a period of time
  following pulsed excitation
• Needs to be chelated requires additional steps
• No polarized emission cannot be used for
  anisotropy measurements

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Transition-Metal-Ligand Complexes

• Ru(II), Re(I), Os(II) with one or more
  dimine ligands
• Display fluorescence from metal-to-ligand
  charge-transfer state, transition forbiddin
  (enhances decay times)

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Protein Sensors

• Zinc sensor zinc finger peptide, Dansyl group
  near middle of the peptide
• Fluorescence enhancement and spectra-shift allow
  zinc detection