lecture 7 Flashcards
What are chromophores?
Molecules that are capable of absorbing visible light or UV light
How are molecules coloured?
Due to chromophores
What must be present in chromophores?
Visible and UV light has low energy so is only sufficient to promote electrons in closely spaced orbitals
What do UV chromophores in proteins provide?
provide delocalised electrons
Where can the delocalised electrons be found?
- conjugated systems
- Transition metals
What does delocalisation of electrons do?
- lowers the energy required to promote electrons
- This allows UV/ visible light be of sufficient energy to cause these promotions
What are Pi bonds?
A covalent chemical bond, formed by sideways overlap of the p orbitals of two carbons atoms of a double bond
What is significance of the peptide bond?
There are delocalised electrons in the peptide bond
Why can there be electron delocalisation in the peptide bond?
Delocalisation of the pi - electrons over the O-C-O , gives a partial double bond character to the C-N bond
Why do transition metals have delocalised electrons?
Transition metals have valence electrons in d orbitals
In fact, these d-orbitals form a sphere of delocalised electrons
What does UV/ visible light do to d - orbital electrons?
UV / visible light causes electrons to gain energy and be promoted to the next level. This delocalisation of electrons lowers the energy required to promote the electrons – so that the low energy of visible and UV light is sufficient to do this.
what does it mean that energy is quantised?
transferred in packets
At what frequency can molecules absorb light if they contain either delocalised electrons or atoms ?
200-800nm
How do these excited electrons return to their ground state?
Vibrational transitions through smaller energy increments
What does absorbed energy appear as?
Heat in solution
What are examples of chromophores in proteins?
- The peptide bond
- Aromatic amino acid side chains
What are UV - visible spectrometers used for?
- can be used to measure the absorbance of ultra violet or visible light by sample , either at a single wavelength or perform a scan over a range in the spectrum
What is the wavelength that the UV- visible spectrometer provides?
200- 800 nm
How is the wavelength measured?
The intensity of light passing through both a reference cell and the sample cell is measured
How is the UV- visible spectrometer plotted?
Plotted as absorbance vs wavelength , the wavelength corresponded to the highest absorption is usually referred to as ‘ lama-max’
Why do more conjugated systems absorbs at less energy?
More conjugation and more delocalisation means that the gap between the pi and pi* has fallen. It takes less energy to excite an electron.
Why would we get two peaks in a UV spectrum?
- PI electrons are In different systems therefore we get two different transitions
What is the most common length of wavelength for amino acid chains to absorb at?
below 200 nm
What wavelength do Phenylalanine, tyrosine and tryptophan have their transitions at?
below 300 nm
What are the uses of UV- visible spectroscopy?
- Quantitative assays
- Structural studies
What are the uses of quantitative assays?
Protein quantification
DNA / RNA quantification
Immunodetection eg. ELISA
Enzymology: rate calculations with absorbant ligands,
What are the uses of structural studies?
Difference spectra can be used to look at changes in folding / assembly / denaturation / ligand binding in different conditions.
What is the Beer - Lambert law?
A = εCL
A = absorbance L = optical path length, i.e. dimension of the cell or cuvette (cm) C = concentration of solution (mol dm-3) ε = molar extinction, which is constant for a particular substance at a particular wavelength (mol-1 cm-1)
What is the Bear - Lambert proportional to?
the absorbance is proportional to the concentration of the substance in solution and as a result UV-visible spectroscopy can also be used to measure the concentration of a sample.
What is the extinction co - efficient?
Extinction coefficient (ε): how strongly a substance absorbs light at a given wavelength, per molar concentration Units: M-1cm-1
What is an example of a protein quantification technique?
Nanodrop UV- visible spectrophotometers
What is the wavelength that is normally monitored?
280 nm is the absorbance used
What are the advantages of protein qualification?
1, It is less dependent on secondary structure
2, There is less interference from some buffers
3, We can calculate extinction coefficients reasonably reliably for most proteins from knowing their sequence.
What is a common protein quantification dye?
BCA
What two reactions foes BCA use?
Peptide bonds in protein reduce Cu2+ ions from the copper(II) sulfate to Cu+
Bicinchoninic acid chelates Cu+, forming a purple-coloured complex that strongly absorbs light at a wavelength of 562nm (detected using spectrophotometer).
What is ELISA?
A form of protein quantification called immunodetection
how does ELISA work?
Antigen / sample added to plate Blocking buffer added Primary antibody added Enzyme-linked secondary antibody added Enzyme substrate added
The enzyme will cleave the substrate and produce a coloured product if the antigen is present.
What happens to haemoglobin that means it can be viewed in a visible spectrum?
- haemoglobin changes colour when it binds to oxygen
- The iron has closely spaced d orbitals
These change their energy level depending on whether a substrate e.g. O2 is bound
The change in energy levels means different wavelengths of light are required to excite the electrons.
What does the UV absorption depend on?
The environment of the chromophore also affects the precise spectrum obtained.
What is difference spectroscopy?
Useful in distinguishing models of protein conformation , following protein denaturation
what is an advantage of difference spectroscopy?
- Able to determine the number of aromatic residues that are exposed to solvent
- Aromatic rings will absorb differently depending on whether they are in water or a hydrophobic environment.
Can be used to study the denaturation of the protein
What is an example of difference spectroscopy?
determining the plane of dissociation in ferrihaemoglobin
Why can haemoglobin dissociate?
Haemoglobin is a tetramer that dissociates into 2 dimers.
Is the interface of dissociation between α1β1 or α1β2 ?
What is the outcome of the plane of dissociation in ferrihaemoglobin ?
- Measure the difference spectra between haemoglobin
- In water (undissociated)
- In 1M NaClO4 (dissociated)
- 2 negative peaks at 292nm and 285nm
- Therefore dissociation occurs along the α1β2 interface
