Spectroscopy Flashcards
Definition
The study of the interaction of matter and electromagnetic radiation.
Western Blotting
Proteins are separated according to their size, and specific antibodies are used to detect how much of our protein of interest is present.
ICC (immunocytochemistry)
Fluorescently tagged antibodies specific to our protein of interest allow visualization with a microscope.
ELISA (enzyme linked immunosorbent assay)
Commonly used to detect antibodies and other proteins in the blood.
Spectrophotometer
Can be used for a protein assay to detect how much general protein is present in a sample
Infrared
Uses bond vibrations to identify chemical bonds and chemical groups present in proteins and sometimes the secondary structure.
Visible and UV
Uses electronic excitation, requires chromophores.
Mainly used for quantification
Fluorescence and FRET
UV light is absorbed and re-emitted at a lower wavelength.
Identifies tertiary and quaternary structures.
Fluoro-microscopy, catalytic studies and FRET is a spectroscopic ruler
Electromagnetic radiation
A type of radiation in which electric and magnetic fields vary simultaneously
Radiation from different parts of the EM spectrum may react _ with biomolecules
Differently
Provides a way to probe the chemical structure of biomolecules
High energy = _ wavelength & _ frequency
Short wavelength, high frequency
Low energy = _ wavelength & _ frequency
Long wavelength and low frequency
λ: Wavelength
The distance from one part of a wave to the corresponding position in the next wave
A: Amplitude
The maximum value that the electric or magnetic vector can have
v: Frequency
The number of times a wave passes through a fixed point in space every second
Unit: Hertz
c: Speed
Unit: m/s
Speed of light: 3 x 10^8 m/s
Equation linking speed, frequency and wavelength
ν = c/λ
Wavenumber
The number of wavelengths per unit distance, typically centimetres (cm−1)
Planck’s Law
E=hv where ν = c/λ
Two waves in phase…
…with the same amplitude and wavelength produce a wave with twice the amplitude and the same wavelength.
Two waves out of phase cancel each other out
Absorption
The light promotes the molecule to an ‘exited state’ and then the energy is released, often in a different form such as heat or light.
Tells you about chemical groups.
Scattering
The light ‘bounces’ off the molecule.
The scattered light has the same wavelength/energy as the incident radiation but changes its direction – shapes.
Biomolecules absorb radiation waves when…
The wavelength of the radiation matches the distance between energy needed to increase to the next energy level.
This is called transition, and involves excitement of electrons, vibrational energy levels in chemical bonds, and also rotational energy levels in single bonds.
Emission spectrum
Detection of the extra energy lost to return to the ground state ∆E
Rotational molecular energy
Energy levels in single covalent bonds between atoms
Vibrational molecular energy
Energy levels in chemical bonds between atoms
Electronic molecular energy levels
Energy levels of electrons within atoms
All molecular energies are _ and have different values
Quantised.
This means they require different radiations to measure them
Near IR region
Quantitative determination of species such as proteins, fats, low-molecular-weight hydrocarbons and water.
Further use in agricultural products, food, petroleum and chemical industries
Mid IR
Most popular of the IR fields, used in determining structures of organic and biochemical compounds
Far IR
Less popular, though it has found uses in inorganic studies.
The electrical output of spectroscopic equipment is _ to the intensity of the radiation beam
Directly proportional
For a vibrational mode in a sample to be IR active it must be…
… associated with changes in the dipole moment
Collimated beam
Produces parallel light rays
Uses of IR
- Identification of functional groups
- Secondary structure information
- Difference spectra
How is IR spectrum used to identify secondary structures
By identifying groups present in those structures, such as hydrogen bonds between amide and carboxyl groups in alpha helices and beta sheets
Difference spectra
Looks at the difference between two spectra acquired in two different states of a protein reaction or of a conformational change
Site directed mutagenesis
This is a comparison of two difference spectra for different proteins in the same conditions.
Chromophores
Molecules or parts of molecules that are capable of absorbing visible light or UV light.
Also responsible for the molecules colour
What causes UV chromophores in proteins?
Delocalised electrons
UV chromophores in proteins
Conjugated systems
Lone pairs on O or N
Transition metals
Chromophores in proteins
The peptide bond (most)
Aromatic amino acid side chains
Uses of quantitative assays
Protein quantification
DNA / RNA quantification
Immunodetection eg. ELISA
Enzymology: rate calculations with absorbant ligands,
Structural studies
Difference spectra can be used to find changes in folding assembly etc in different conditions
Beer-Lambert law
A=εCL
* A = absorbance
* L = optical path length
* C = concentration of solution
* ε = molar extinction (how strongly a substance absorbs light at a given wavelength)
Why do we normally measure absorbance of side chains at 280nm
It’s less dependent on secondary structure and there is less interference from some buffers, allowing us to calculate coefficients reliably
Example wavelengths used
- 280nm: aromatic side chains
- 205nm: for proteins without aromatic side chains (peptide bond)
- 260nm: DNA and RNA
(can distinguish dsDNA, SSDNA and RNA as they have different extinction coefficients)
BCA protein assay uses two reactions
Peptide bonds in protein reduce Cu2+ ions from the copper sulfate to Cu+
Bicinchoninic acid chelates Cu+, forming a purple-coloured complex that strongly absorbs light at a wavelength of 562nm
If Beer-Lambert is obeyed, absorbance vs concentration is _
Linear
Steps of ELISA
- Antigen / sample added to plate
- Blocking buffer added
- Primary antibody added
- Enzyme-linked secondary antibody added
- Enzyme substrate added
Purpose of ELISA
Immunodetection
What is a positive result for ELISA?
Coloured product.
The enzyme will cleave the substrate and produce a coloured product if the antigen is present.
Uses of difference spectroscopy
Determining protonation state of side chains
pKa values
Structural analyses - tertiary and quaternary
Which amino acid fluoresces?
Tryptophan
What is fluorescence?
When UV light is absorbed and re-emitted at a longer wavelength.
Common uses for Fluorescence spectroscopy
- 3 and 4 structures
- Measuring distances
- Catalytic studies
- Fluorescence microscopy
Why does Fluorescence occur?
Excitation energy excites electron to a higher energy state.
Some rigid, inflexible chromophores can’t lose that excess energy as vibrations so it is lost as radiation or light.
High energy = _er wavelength
Shorter
Biophosphorescence
Needs initial light source to activate, then continues to “glow”
Bioluminescence
No need for activating light source, chemicals release light on their own.
Properties of a fluorophore
- must be a chromophore
- rigid structure
- short lifetime of the excited state (<10^-9 seconds)
Extinction coefficient
Indicates how much light they absorb
What is Q?
A measure of how much absorbance is turned into fluorescence.
1 is the max, but 0.1 is still quite fluorescent
Q is affected by:
Internal factors - distribution of energy levels etc
External factors - quenching
External fluors
We label a protein with fluorescent tags (GFP usually) to enable detection.
Protein cloned into a vector so that when expressed it is attached to fluorophore.
Spectrofluorimeter
- Incident beam of radiation of a given wavelength is passed through a sample cuvette containing the fluor.
- Emitted radiation (of longer wavelength) is detected by a photomultiplier tube
- Two monochromators in this system:
- The emitted radiation is detected at 90 degrees to the direction of the incident light beam
Why are there two monochromators in the spectrofluorimeter?
o One to select wavelength of light required for excitation
o One to select for the wavelength of emitted light
Uses of fluorescent spectroscopy
- Structural studies – tertiary and quaternary protein structure
- FRET: Measuring distances within proteins and complexes
- Binding / catalytic studies using a fluorescent substrate
- Fluorescence microscopy
Environments that might effect emission of trp
- Solvent polarity (exposure to aqueous phase)
- Proximity of protonated groups such as Asp or Glu
- Quenching by iodine, acrylamide and nearby disulphide groups
- Quenching by nearby electron deficient groups like –NH3, -CO2H and protonated histidine residues
Wavelength of trp buried in tetramer
Shorter
Wavelength of trp on surface of monomer
Longer
FRET
Studies energy transferred between fluors via resonance.
Can occur when emission spec of one fluor overlaps with another
Works best across 10-80A
Uses of FRET
Measures the distances within proteins
Measure by comparison of fluorescence of acceptor fluor
Can do this because the efficiency of electron transfer is related to the distance between the fluors.
Efficiency of electron transfer in FRET depends on:
- The distance between the donor and acceptor
- The spectral overlap of the donor emission spectrum and the acceptor absorption spectrum
- The relative orientation of the donor emission dipole moment and the acceptor absorption dipole moment
Ligand binding / catalytic studies
The ligand or substrate is fluorescently labeled.
When receptor-ligand binding or enzyme-substrate binding occurs, there is a change in fluorescence.
What can you calculate from ligand binding kinetics?
Kd and receptor affinites
Fluorescence microscopy
- A labelled (fluorophore or colour) secondary antibody binds to the primary antibody, and allows visualisation by microscopy
- The fluorescent tag (e.g. GFP / FITC) on the antibody is excited by the specified wavelength of light from the light source, which is directed to the sample
- The emitted light travels to the eyepiece (and camera) which is photographed.
Right and left circularly polarised light interacts differently with _ chromaphores
Chiral
What do chiral chromaphores do to circularly polarised light?
THey cause it to become elliptically polarised
Circular dichroism
Use of circular dichroism spectroscopy
Secondary structure elucidation
Plane polarised light
light travelling on only one direction
Electric vector direction is constant
Filters for this are made of long organic chains.
Elliptically polarised light
Caused by a molecule differentially absorbing L & R beans so that they have different amplitudes
Properties of a substance that can circular dichroism
Ability to absorb light (chromophore)
Asymmetric / contain CHIRAL residues
Therefore it must either:
* Contain a peptide bond
* Contain aromatic residues in asymmetric environments
* Contain DNA bases in asymmetric environments
Most amino acids are _-enantiomers
L
Most monosaccharides are _-enantiomers
D
L-amino acids result in _ handed alpha helices in proteins
Right
L vs D chirality
L: Laevorotation (rotates to the left)
D: Dextrorotation (rotates to the right)
Ellipticity
Unit of CD
The tangent of the ratio of the minor to major elliptical axis
Units of millidegrees
If a molecule contains a chiral chromophore, the CD signal will be _
Non-zero
If left CPL is absorbed more than right CPL, the CD signal is _
Positive
How to increase accuracy?
Use more data points
Uses of circular dichroism spectroscopy
- Protein secondary structure determination
- Monitoring changes in protein structure
- Nucleic acid structure
- Protein-protein and protein-DNA interactions
Circular Dichroism spectroscopy is an example of a _ spectra
Difference
The CD signal for a protein depends on its _ structure
Secondary
What wavelength is used for nucleotides in CD?
Near UV - above 240nm
What wavelength is used to measure changes in protein secondary structure following binding to DNA?
Far UV - below 240nm
