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BC3009: Biophysical & Biochemical Methods > Chromophores > Flashcards

Chromophores Flashcards

1
Q

What is a Chromophore?

A

Molecular structure which interacts with photons

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2
Q

What are the properties of a Chromophore?

A
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3
Q

Give an example of some Chromophores

A
  • Vitamin A - Abs. = 450 nm (dark blue), Emission is Yellow
  • Beta Carotene - Abs. = 475 nm (light blue), Emission is Orange
  • Chlorophyll a - Absorbance is Red/Blue, Emission is Green
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4
Q

Explain chromophores with respect to colour

A
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5
Q

Explain chromophores with respect to HOMO and LUMO

A
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6
Q

Why is Beta Carotene orange?

A

Conjugated system spreads over 11 carbon to carbon double bonds that reduces the gap between HOMO and LUMO so that the light absorbed is light blue range (475 nm)

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7
Q

What is Bathochromic shift?

A
  • Red shift
  • Involves shift of absorption max. towards longer wavelength
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8
Q

What is Hypsochromic shift?

A
  • Blue shift
  • Shift of a band to higher energy or shorter wavelength
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9
Q

What is Hyperchromic shift?

A

Increase in the molar absorptivity

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10
Q

What is Hypochromic shift?

A

Decrease in the molar absorptivity

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11
Q

What is the relationship between conjugation and bathochromic or hyper chromic shift?

A

The higher the conjugation the higher the bathochromic shift

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12
Q

Name THREE types of chromophores in proteins

A
  1. Peptide bonds - 210nm
  2. AA side chains (Y&W) - 280nm
  3. Prosthetic Haem group - 400nm
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13
Q

Explain the chromophore nature of FAD

A

As FAD becomes more reduced its absorption at 450nm reduces

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14
Q

What would the graph look like for the absorption of FAD, FADH and FADH2 across a 350nm to 550nm wavelength range?

A
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15
Q

Explain the chromophore nature of NAD

A
  • The reduced form of NAD, NADH, absorbs at 339nm
  • The oxidised form, NAD+, does not
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16
Q

Explain how NAD(P) dehydrogenase enzymatic activity could be assayed using spectrophotometry

A

The activity of dehydrogenase can be tested by analysing the way absorption levels go up as dehydrogenase reduces NAD+ to NADH

17
Q

Explain G6PD deficiency and the spectroscopic method used for its diagnosis?

A
  • Highly prevalent - 400 million people affected worldwide
  • Quantitative spectrophotometric assay used to detect G6PD activity
  • Based on 340 nm absorption given by rate of NADPH formation
  • Precise amount of hemolysate added to assay mixture containing substrate (glucose-6-phosphate) and cofactor NADP
18
Q

Explain the biochemistry of the G6PD spectrophotometric assay and its application for screening for G6PD deficiency

A
  • Glucose-6-Phosphate converted to a product by G6PD
  • NADPH molecule formed from NADP+
  • Rate at which NADP+ is converted to NADPH is proportional to G6PD activity
19
Q

Explain how G6PD is quantitated using spectrophotometry

A

G6PD activity expressed as:
G6PD IU/RBCs and G6PD IU/Hemoglobin ratio

20
Q

What does 1 IU of G6PD mean?

A

1 International Unit or 1µmol of substrate converted per minute by G6PD

21
Q

Why is G6PD values shown as IU/gHb?

A
  • Hb and G6PD are both found in RBCs
  • gHb is related to number of RBCs in a sample and amount of available G6PD enzyme
  • The HIGHER the gHb, the HIGHER the number of RBCs and amount of G6PD available
22
Q

Explain how G6PD inhibitor Wedelolactone was identified

A
  • NADPH absorption photometry assay to characterise G6PD activity
  • High-throughput screening (HTS) to an in-house library performed
  • Wedeloactone compound identified that inhibits G6PD strongly in a non-competitive, reversible way
23
Q

Explain IC50 and EC50

A
  • IC50 is the concentration of an inhibitor where the response (or binding) is reduced by half
  • EC50 is the concentration of a drug that gives half-maximal response
24
Q

Explain the coupled enzyme assay to measure the activity of lactate dehydrogenase

A
  • Lactate dehydrogenase (LDH) activity can be quantified using 340nm absorption photometry of NADH
  • NADH produces poor signals and interfered with by other molecules
  • Diaphorase uses NADH to convert Tetrazolium salt to Formazam, which produces a colour change from blue to red
  • The colour change can be detected using 490 nm which is less susceptible to interference and produces a stronger signal than 340nm NADH
25
Q

Explain how is the enzyme diaphorase used in a lactate dehydrogenase assay

A
  • Uses NADH to convert Tetrazolium salt to Formazam
  • Produces a red colour whch is related to the amount of NADH available to Diaphorase
  • NADH is produced by LDH proportionally to conversion of enzyme
  • Activity of LDH is related to 490nm absorption of Formazam as LDH produces NADH
  • Diaphorase uses NADH to convert Tetrazolium to Formazam
26
Q

Explain how chromophores can be used in enzyme assays and give and example of an enzyme assay that uses a chromophore

A

BC3009: Biophysical & Biochemical Methods (12 decks)

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