lecture 7

Question 1

What are chromophores?

Answer 1

Molecules that are capable of absorbing visible light or UV light

Question 2

How are molecules coloured?

Answer 2

Due to chromophores

Question 3

What must be present in chromophores?

Answer 3

Visible and UV light has low energy so is only sufficient to promote electrons in closely spaced orbitals

Question 4

What do UV chromophores in proteins provide?

Answer 4

provide delocalised electrons

Question 5

Where can the delocalised electrons be found?

Answer 5

• conjugated systems

- Transition metals

Question 6

What does delocalisation of electrons do?

Answer 6

• lowers the energy required to promote electrons

- This allows UV/ visible light be of sufficient energy to cause these promotions

Question 7

What are Pi bonds?

Answer 7

A covalent chemical bond, formed by sideways overlap of the p orbitals of two carbons atoms of a double bond

Question 8

What is significance of the peptide bond?

Answer 8

There are delocalised electrons in the peptide bond

Question 9

Why can there be electron delocalisation in the peptide bond?

Answer 9

Delocalisation of the pi - electrons over the O-C-O , gives a partial double bond character to the C-N bond

Question 10

Why do transition metals have delocalised electrons?

Answer 10

Transition metals have valence electrons in d orbitals

In fact, these d-orbitals form a sphere of delocalised electrons

Question 11

What does UV/ visible light do to d - orbital electrons?

Answer 11

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.

Question 12

what does it mean that energy is quantised?

Answer 12

transferred in packets

Question 13

At what frequency can molecules absorb light if they contain either delocalised electrons or atoms ?

Answer 13

200-800nm

Question 14

How do these excited electrons return to their ground state?

Answer 14

Vibrational transitions through smaller energy increments

Question 15

What does absorbed energy appear as?

Answer 15

Heat in solution

Question 16

What are examples of chromophores in proteins?

Answer 16

• The peptide bond

- Aromatic amino acid side chains

Question 17

What are UV - visible spectrometers used for?

Answer 17

• 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

Question 18

What is the wavelength that the UV- visible spectrometer provides?

Answer 18

200- 800 nm

Question 19

How is the wavelength measured?

Answer 19

The intensity of light passing through both a reference cell and the sample cell is measured

Question 20

How is the UV- visible spectrometer plotted?

Answer 20

Plotted as absorbance vs wavelength , the wavelength corresponded to the highest absorption is usually referred to as ‘ lama-max’

Question 21

Why do more conjugated systems absorbs at less energy?

Answer 21

More conjugation and more delocalisation means that the gap between the pi and pi* has fallen. It takes less energy to excite an electron.

Question 22

Why would we get two peaks in a UV spectrum?

Answer 22

• PI electrons are In different systems therefore we get two different transitions

Question 23

What is the most common length of wavelength for amino acid chains to absorb at?

Answer 23

below 200 nm

Question 24

What wavelength do Phenylalanine, tyrosine and tryptophan have their transitions at?

Answer 24

below 300 nm

Question 25

What are the uses of UV- visible spectroscopy?

Answer 25

- Quantitative assays | - Structural studies

Question 26

What are the uses of quantitative assays?

Answer 26

Protein quantification DNA / RNA quantification Immunodetection eg. ELISA Enzymology: rate calculations with absorbant ligands,

Question 27

What are the uses of structural studies?

Answer 27

Difference spectra can be used to look at changes in folding / assembly / denaturation / ligand binding in different conditions.

Question 28

What is the Beer - Lambert law?

Answer 28

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) ```

Question 29

What is the Bear - Lambert proportional to?

Answer 29

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.

Question 30

What is the extinction co - efficient?

Answer 30

``` Extinction coefficient (ε): how strongly a substance absorbs light at a given wavelength, per molar concentration Units: M-1cm-1 ```

Question 31

What is an example of a protein quantification technique?

Answer 31

Nanodrop UV- visible spectrophotometers

Question 32

What is the wavelength that is normally monitored?

Answer 32

280 nm is the absorbance used

Question 33

What are the advantages of protein qualification?

Answer 33

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.

Question 34

What is a common protein quantification dye?

Answer 34

BCA

Question 35

What two reactions foes BCA use?

Answer 35

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).

Question 36

What is ELISA?

Answer 36

A form of protein quantification called immunodetection

Question 37

how does ELISA work?

Answer 37

``` 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.

Question 38

What happens to haemoglobin that means it can be viewed in a visible spectrum?

Answer 38

- 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.

Question 39

What does the UV absorption depend on?

Answer 39

The environment of the chromophore also affects the precise spectrum obtained.

Question 40

What is difference spectroscopy?

Answer 40

Useful in distinguishing models of protein conformation , following protein denaturation

Question 41

what is an advantage of difference spectroscopy?

Answer 41

- 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

Question 42

What is an example of difference spectroscopy?

Answer 42

determining the plane of dissociation in ferrihaemoglobin

Question 43

Why can haemoglobin dissociate?

Answer 43

Haemoglobin is a tetramer that dissociates into 2 dimers. | Is the interface of dissociation between α1β1 or α1β2 ?

Question 44

What is the outcome of the plane of dissociation in ferrihaemoglobin ?

Answer 44

- 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