9.1 UV-visible and fluorescence spectroscopy

UV-visible and fluorescence spectroscopy are powerful tools for studying biomolecules. These techniques use light to probe electronic transitions and excited states, revealing crucial information about structure, interactions, and dynamics of proteins and nucleic acids.

Both methods offer unique advantages in biophysical research. UV-visible spectroscopy measures light absorption, while fluorescence detects emitted light. Together, they provide insights into molecular properties, conformational changes, and binding events, complementing other spectroscopic techniques in the biophysicist's toolkit.

Principles of UV-Visible Spectroscopy

Absorption of Light and Electronic Transitions

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• UV-visible spectroscopy is based on the absorption of ultraviolet and visible light by molecules, causing electronic transitions between molecular orbitals
• The energy of the absorbed light corresponds to the energy difference between the ground state and excited state of the molecule, following the Bohr frequency condition ($ΔE = hν$)
• The absorption of light by a sample is governed by the Beer-Lambert law, which relates the absorbance ($A$) to the concentration ($c$) of the absorbing species, the path length ($l$) of the sample, and the molar extinction coefficient ($ε$) of the molecule ($A = εlc$)
  • The molar extinction coefficient is a measure of the probability of the electronic transition and is characteristic of the chromophore (light-absorbing group) in the molecule

Applications in Studying Biomolecules

• Proteins and nucleic acids absorb UV light due to the presence of aromatic amino acids (tryptophan, tyrosine, and phenylalanine) and nucleobases (adenine, guanine, cytosine, thymine, and uracil), respectively
• UV-visible spectroscopy can be used to determine the concentration of biomolecules in solution using the Beer-Lambert law and the known extinction coefficients of the chromophores
• Changes in the UV-visible spectrum of a biomolecule can provide information about its conformational changes, ligand binding, or denaturation, as these events can alter the electronic environment of the chromophores
  • For example, the binding of a ligand to a protein may cause a shift in the absorption maximum or a change in the intensity of the absorption band

Fluorescence in Biophysical Studies

Principles of Fluorescence

• Fluorescence is the emission of light by a molecule that has absorbed light of a higher energy (shorter wavelength)
  • The emitted light has a lower energy (longer wavelength) than the absorbed light due to vibrational relaxation and solvent reorganization
• The difference between the absorption and emission maxima is called the Stokes shift, which is influenced by the solvent polarity and the molecular structure of the fluorophore
• The fluorescence quantum yield ($Φ$) is the ratio of the number of photons emitted to the number of photons absorbed
  • It is a measure of the efficiency of the fluorescence process and is affected by competing non-radiative processes, such as intersystem crossing and collisional quenching
• The fluorescence lifetime ($τ$) is the average time a molecule spends in the excited state before returning to the ground state by emitting a photon
  • It is sensitive to the local environment of the fluorophore and can be affected by quenching processes

Fluorescence-based Techniques in Biophysics

• Intrinsic fluorophores in biomolecules include aromatic amino acids (tryptophan, tyrosine, and phenylalanine) and enzyme cofactors (NADH and FAD)
  • Extrinsic fluorophores, such as fluorescein and rhodamine, can be covalently attached to biomolecules for specific labeling
• Fluorescence spectroscopy can be used to study protein folding, conformational dynamics, ligand binding, and enzyme kinetics by monitoring changes in the fluorescence intensity, wavelength, anisotropy, or lifetime of the fluorophores
• Förster resonance energy transfer (FRET) is a fluorescence-based technique that can measure the distance between two fluorophores (donor and acceptor) in the range of 1-10 nm
  • FRET is useful for studying biomolecular interactions and conformational changes, such as protein-protein interactions or the folding of a protein domain

Interpreting Spectra for Biomolecular Insights

UV-Visible Spectroscopy

• The shape and intensity of the UV-visible absorption spectrum can provide information about the secondary structure of proteins
  • For example, α-helical proteins exhibit a characteristic negative peak at 222 nm and a positive peak at 190 nm in circular dichroism (CD) spectroscopy
• The red shift (bathochromic shift) or blue shift (hypsochromic shift) of the absorption or emission maxima can indicate changes in the polarity of the microenvironment around the chromophore or fluorophore, which may occur during protein folding or ligand binding
  • A red shift suggests a more polar environment, while a blue shift indicates a more hydrophobic environment

Fluorescence Spectroscopy

• The fluorescence emission spectrum of tryptophan is sensitive to its local environment
  • In a hydrophobic core of a protein, tryptophan exhibits a blue-shifted emission maximum compared to its emission in an aqueous solution
• Quenching of fluorescence can occur due to various mechanisms, such as collisional quenching, static quenching, or FRET
  • Analyzing the Stern-Volmer plot ($F_0/F$ vs. $[Q]$) can help distinguish between dynamic and static quenching and determine the accessibility of the fluorophore to the quencher
• The fluorescence anisotropy ($r$) measures the rotational diffusion of the fluorophore, which depends on the size and shape of the molecule
  • Changes in anisotropy can indicate protein-ligand binding, protein-protein interactions, or conformational changes that affect the rotational mobility of the fluorophore
• Time-resolved fluorescence measurements, such as fluorescence lifetime and time-correlated single photon counting (TCSPC), can reveal the heterogeneity of the fluorophore's environment and the presence of multiple conformational states

UV-Visible vs Fluorescence Spectroscopy

Advantages and Limitations

• Advantages of UV-visible spectroscopy:
  • Simple, fast, and non-destructive technique
  • Requires small sample volumes and low concentrations
  • Can be used for quantitative analysis using the Beer-Lambert law
  • Provides information about the electronic structure and conformational changes of biomolecules
• Limitations of UV-visible spectroscopy:
  • Limited structural information compared to other techniques like NMR or X-ray crystallography
  • Interference from other absorbing species in complex mixtures
  • Difficulty in assigning specific absorption bands to individual chromophores in proteins
• Advantages of fluorescence spectroscopy:
  • High sensitivity and specificity, allowing detection of low concentrations of fluorophores
  • Can provide information about the local environment, conformational dynamics, and interactions of biomolecules
  • Suitable for studying kinetic processes in real-time
  • Multiplexing capabilities using different fluorophores with distinct excitation and emission spectra
• Limitations of fluorescence spectroscopy:
  • Requires the presence of fluorophores, which may be intrinsic or need to be introduced by labeling
  • Labeling with extrinsic fluorophores may perturb the native structure or function of the biomolecule
  • Photobleaching of fluorophores can limit the observation time and signal intensity
  • Inner filter effects, self-quenching, and background fluorescence can interfere with the measurements

Comparison with Other Biophysical Techniques

• UV-visible and fluorescence spectroscopy provide complementary information to other techniques, such as circular dichroism (CD), infrared (IR) spectroscopy, nuclear magnetic resonance (NMR), and X-ray crystallography
  • CD spectroscopy provides information about the secondary structure of proteins and nucleic acids
  • IR spectroscopy can probe the vibrational modes of biomolecules and provide information about their functional groups and hydrogen bonding
  • NMR spectroscopy offers high-resolution structural and dynamic information about biomolecules in solution
  • X-ray crystallography provides detailed three-dimensional structures of biomolecules in the solid state
• The choice of technique depends on the specific research question, sample properties, and available resources
  • UV-visible and fluorescence spectroscopy are often used as initial screening methods or for monitoring changes in biomolecular systems, while more advanced techniques like NMR and X-ray crystallography are employed for in-depth structural characterization