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BB10186: Proteins purification and characterisation > lecture 7 > Flashcards

lecture 7 Flashcards

1
Q

What are chromophores?

A

Molecules that are capable of absorbing visible light or UV light

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2
Q

How are molecules coloured?

A

Due to chromophores

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3
Q

What must be present in chromophores?

A

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

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4
Q

What do UV chromophores in proteins provide?

A

provide delocalised electrons

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5
Q

Where can the delocalised electrons be found?

A
  • conjugated systems

- Transition metals

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6
Q

What does delocalisation of electrons do?

A
  • lowers the energy required to promote electrons

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

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7
Q

What are Pi bonds?

A

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

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8
Q

What is significance of the peptide bond?

A

There are delocalised electrons in the peptide bond

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9
Q

Why can there be electron delocalisation in the peptide bond?

A

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

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10
Q

Why do transition metals have delocalised electrons?

A

Transition metals have valence electrons in d orbitals

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

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11
Q

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

A

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.

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12
Q

what does it mean that energy is quantised?

A

transferred in packets

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13
Q

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

A

200-800nm

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14
Q

How do these excited electrons return to their ground state?

A

Vibrational transitions through smaller energy increments

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15
Q

What does absorbed energy appear as?

A

Heat in solution

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16
Q

What are examples of chromophores in proteins?

A
  • The peptide bond

- Aromatic amino acid side chains

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17
Q

What are UV - visible spectrometers used for?

A
  • 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
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18
Q

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

A

200- 800 nm

19
Q

How is the wavelength measured?

A

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

20
Q

How is the UV- visible spectrometer plotted?

A

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

21
Q

Why do more conjugated systems absorbs at less energy?

A

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

22
Q

Why would we get two peaks in a UV spectrum?

A
  • PI electrons are In different systems therefore we get two different transitions
23
Q

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

A

below 200 nm

24
Q

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

A

below 300 nm

25
Q

What are the uses of UV- visible spectroscopy?

A
  • Quantitative assays

- Structural studies

26
Q

What are the uses of quantitative assays?

A

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

27
Q

What are the uses of structural studies?

A

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

28
Q

What is the Beer - Lambert law?

A

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)
29
Q

What is the Bear - Lambert proportional to?

A

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.

30
Q

What is the extinction co - efficient?

A 
Extinction coefficient (ε): how strongly a substance absorbs light at a given wavelength, per molar concentration
Units: M-1cm-1
31
Q

What is an example of a protein quantification technique?

A

Nanodrop UV- visible spectrophotometers

32
Q

What is the wavelength that is normally monitored?

A

280 nm is the absorbance used

33
Q

What are the advantages of protein qualification?

A

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.

34
Q

What is a common protein quantification dye?

A

BCA

35
Q

What two reactions foes BCA use?

A

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).

36
Q

What is ELISA?

A

A form of protein quantification called immunodetection

37
Q

how does ELISA work?

A 
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.

38
Q

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

A
  • 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.
39
Q

What does the UV absorption depend on?

A

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

40
Q

What is difference spectroscopy?

A

Useful in distinguishing models of protein conformation , following protein denaturation

41
Q

what is an advantage of difference spectroscopy?

A
  • 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

42
Q

What is an example of difference spectroscopy?

A

determining the plane of dissociation in ferrihaemoglobin

43
Q

Why can haemoglobin dissociate?

A

Haemoglobin is a tetramer that dissociates into 2 dimers.

Is the interface of dissociation between α1β1 or α1β2 ?

44
Q

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

A
  • 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

BB10186: Proteins purification and characterisation (10 decks)

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