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Biochemistry 2 > Staniforth > Flashcards

Staniforth Flashcards

1
Q

What must be recorded to measure reaction rates/binding constants?

A

The amounts of product and/or reactants quantitatively as a function of time

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2
Q

What does quantitatively mean?

A
  • Quantitative information or data is based on quantities obtained using a quantifiable measurement process
  • Numerical data
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3
Q

What does qualitative mean?

A

Qualitative means relating to the nature or standard of something, rather than to its quantity

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4
Q

What is direct measurement?

A

Whereby the reaction is observed directly as it happens

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5
Q

What are non-invasive methods?

A

Methods that do not affect the course of the reaction, so a single reaction mixture is used throughout

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6
Q

What are the limitations of direct measurement?

A

It is rare to be able to monitor all components of the reaction simultaneously and the type of reactions that can be measured directly are limited

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7
Q

What are the advantages of direct measurement?

A

They are simpler and quicker than quenching methods. There are fewer steps which means there is smaller error and better signal to noise

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8
Q

How are direct measurement reactions probed?

A
  • By using techniques that are specific to the chemical of interest
  • A signal from the product/ other component such as the buffer should not cloud that of the substrate
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9
Q

What are quenching methods?

A

Reaction is arrested and then the extent of the reaction is assessed at each time point

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10
Q

How is the reaction set up in quenching methods?

A
  • A separate reaction mixture is set up for each timepoint

- Or (more commonly) an aliquot from the reaction mixture is taken and mixed with quenching agent

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11
Q

What are the advantages of quenching methods?

A

A separate instrument is used for the detection and quantification of the chemicals of interest

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12
Q

What are the three stages to quenching methods?

A
  1. Starting (triggering)
  2. Stopping (quenching)
  3. Separation of components of reaction (separation of products and substrates)
  4. Quantification/identification
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13
Q

What does separation of components in quenching methods allow for?

A

Makes it possible to use non-specific probes to measure amounts of substrate and product

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14
Q

What factors affect the choice of methodology for assaying rates of biochemical reactions?

A
  1. Range of applicability
  2. Timescale
  3. Sensitivity
  4. Availability
  5. Accuracy
  6. Cost
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15
Q

How does range of applicability affect choice of methodology?

A

Many methods require specific properties in their chemical they can monitor

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16
Q

What types of biological probes can be used?

A
  • ADP/ATP - Radioactive
  • DNA - Fluorescent, Absorbant, radioactive and can react with fluorescent dyes
  • Glucose - Not a good probe
  • Haem-Absorbant and fluroescent (depending on ligand bound and oxidation)
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17
Q

How does timescale affect the choice of methodology for a biochemical assay?

A
  • Biochemical reactions occur at many different timescales from seconds to billions of years, this affects how they are measured and what techniques can be used e.g. NMR is very slow whereas stopped flow detected by absorbance etc is as quick as 5 milliseconds
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18
Q

How can sensitivity affect a biochemical assay method?

A
  • Biological reactions are carried at a range of different substrate concentrations
  • Depending on what you measure, working at high or low concentrations may affect the methodology that you use
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19
Q

How can availability affect a biochemical assay method?

A

Many things are now possible, however resources and availability of more sophisticated equipment may affect your choice

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20
Q

How can accuracy affect your choice of biochemical assay method?

A

Some methods give more reliable results than others

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21
Q

How can cost affect your choice of biochemical assay method?

A
  • Some technology is prohibitively expensive
  • Therefore cheaper techniques are often used for initial characterisation and more complex techniques are used if and when necessary
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22
Q

How can short timescale affect mixing?

A
  • If reaction occurs very fast, mixing needs to be carried out very fast
  • Manual mixing is not very accurate for reactions with a timescale below 1 minute
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23
Q

What is required to trigger a reaction?

A

Mixing together reactants, enzymes and cofactors

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24
Q

What methods are used if mixing cannot be done fast enough?

A

If mixing cannot be done fast enough, equilibrium perturbation methods are used

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25
Q

What is equilibrium perturbation?

A

These techniques do not mix two solutions, instead they rely on other factors to trigger the start of a reaction

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26
Q

What is an example of an equilibrium perturbation technique?

A
  1. At a low temp, reaction doesn’t occur as position of equilibrium doesn’t favour reaction.
  2. Upon heating, equilibrium shifts to a new position where the reaction can occur and the reaction is observed using standard spectroscopic techniques
    - This is because the temp jump and pressure jump apparatus changes the position of the equilibrium in a reaction
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27
Q

What technique in quenching methods is often used to separate components of reaction?

A
  • HPLC is a modern favourite
  • It places separation technology (chromatography) directly upstream of a wide variety of detectors. This increases accuracy of the technology greatly
  • Old methods are the main ones used though
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28
Q

What signal of choice is often used when sensitivity is required?

A

Radioactivity

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29
Q

If sensitivity is not required, what other signals of choice are used?

A

Fluorescence or absorbance

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30
Q

What are direct (non-invasive methods) used?

A

Spectroscopies:

  1. Absorbance
  2. Fluorescence
  3. pH meter/pH stat
  4. SPR (Surface plasmon resonance)
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31
Q

Where do biological molecules tend to be optically active?

A

In the UV visible region of the spectrum

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32
Q

What electromagnetic wavelength to spectrophotometers often use?

A

UV light (400-800nm) but most molecules range 190-750nm

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33
Q

What is NMR sensitive to and what can this be harnessed for?

A

Very sensitive to chemical environment of Hydrogen (H1), Nitrogen (N15), and Carbon (C13) and therefore can be used for simple quantitative measurements of small organic compounds as well as highly complex determination of protein structures

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34
Q

What are x-rays often used for?

A

To explore the structure of macromolecules in crystals

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35
Q

What is the frequency of electromagnetic radiation proportional to?

A

The energy and the wavelength

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36
Q

What are the stages of absorbance?

A
  1. A light beam passes through a prism and the wavelength(s) of interest are selected using a monochromator
  2. The monochromator produces incident light which is shined onto the sample
  3. The transmitted light is then detected using a photomultiplier tube which transforms observed photons into an electrical signal read by the computer
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37
Q

What equation/law is used for absorbance?

A

Beers law

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38
Q

What is Beer’s Law?

A

Used to establish a relationship between concentration and absorbance in photometric determinations

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39
Q

What is the Beer’s Law equation?

A

Beers law = logi0 - Logi

40
Q

What does the observed absorbance equation tell you?

A

That measured signal (absorbance) is directly proportional to the concentration of the absorbing compound

41
Q

What is fluorescence?

A

A property of certain compounds that after absorbing light at one wavelength, emit light at another wavelength

42
Q

What does absorption refer to in fluorescence?

A

The excitation of the fluorophore

43
Q

What does emitted often refer to in fluorescence?

A

The light emitted is the fluorescence emission

44
Q

What does the fluorescence machine contain in order?

A

Lamp –> Monochromator –> Excitation of sample –> Emission –> Monochromator –> Photo-multiplier –> Detector

45
Q

What is fluorescence proportional to?

A

Concentration (therefore quantitative)

46
Q

What type of reactions is a pH meter/stat used for?

A

Reactions involving the reaction/production of an acid

47
Q

What can a pH meter be used to measure?

A

The disappearance of substrate or the appearance of product

48
Q

What does a pH stat contain?

A
  • pH meter with electrode in sample
  • Stirrer of sample
  • Reaction vessel containing sample
  • Pump which pumps in NaOH or HCl
49
Q

What is the observed signal for a pH meter?

A

The volume of added acid or base

50
Q

What is the advantage of Surface plasmon resonance?

A

It is label free and therefore it does not require the reaction to have an optical signal

51
Q

What does SPR stand for?

A

Surface Plasmon Resonance

52
Q

What is measured in SPR?

A

SPR determines the dissociation constant (Kd) for a ligand binding protein

53
Q

What can SPR NOT measure?

A

RATES OF REACTION

54
Q

How is Kd calculated in SPR?

A

Kd = Koff/Kon

55
Q

What does k off and k on refer to in SPR?

A

K off = backwards reaction (LP—> L+P)

K on = forward reaction (L+P–>LP)

56
Q

What is are the principles of SPR?

A
  1. Ligand is attached to a polymer immobilised onto a solid surface
  2. A solution of protein flows through over this surface and binding is measured as a change in refractive index (property of all compounds to bounce [reflect] off light)
  3. This method is highly sensitive and is dependent on the mass bound
57
Q

What is SPR dependent on?

A

The mass of ligand bound to protein which causes a change in the angle of light refracted off solid surface

58
Q

In SPR what is the change in angle of refracted light proportional to?

A

The mass bound to the solid surface

59
Q

What are the disadvantages of SPR?

A

Absolute value of binding of ligand to protein can be underestimates as binding on surfaces is not always perfect mimic of what occurs in bulk solution

60
Q

When/Who is SPR used by?

A

SPR is used a lot by pharmacologists screening for drugs to bind a protein target

61
Q

What is SPR good at suggesting in terms of pharmacology?

A

Which drugs binds tightest although hit may not provide an accurate determination of the real Kd

62
Q

What is continuous flow?

A

Very old technology (only recently been developed to measure fast reactions occurring over 10micro seconds) - DEAD TIME APPARATUS

63
Q

What is the principle of continuous flow?

A
  • To push 2 liquids into a small capillary using pneumatically driven ram (pushing) at a constant rate
  • At the entrance to the observation cell there is a platinum ball which serves as a mixer (pushing liquids against it causes enough turbulence to mix the solutions FAST)
64
Q

What does the platinum ball serve as in the entrance to the observation cell in continuous flow?

A

As a mixer, pushing liquids against it causes enough turbulence to mix the solutions FAST

65
Q

How big is the observation cell in continuous flow?

A

1cm long but only 050um (o.25mm) wide

66
Q

How is data collected in continuous flow?

A

Light is shone on the length of the observation cell and data is collected using a special CCD detector

67
Q

What does a CCD detector in continuous flow do?

A

It resolves the length of the observation cell into time points

68
Q

What is the history of a stopped flow apparatus?

A

Developed successfully in 1940s and is now available commercially

69
Q

What is the dead time of a stopped flow apparatus?

A
  • Fast mixing device

- 1x10-3 seconds (1ms)

70
Q

What are fast mixing techniques?

A
  • Continuous flow
  • Stopped flow
  • Equilibrium perturbation techniques
71
Q

What does equilibrium perturbation exploit?

A

The use of infrared lasers to cause rapid changes in the temperature which means that in order to cause a temperature jump of the order of 5 to 10 degrees, the dead time of these machines is in the range of 10-100x10-9 seconds (10-100ns)

72
Q

What is the dead time of equilibrium perturbation techniques?

A

10-100ns

73
Q

What are the limitations of continuous flow?

A

You cannot use it unless the reaction is EXTREMELY FAST

74
Q

What is flash photolysis?

A

Use of lasers to break specific chemical bonds in specially designed compounds such as ‘caged ATP’

75
Q

What happens upon flash photolysis?

A

On irradiation with a laser pulse, ‘caged ATP’ releases ATP into the solution and the reaction is thus triggered

76
Q

What is the timescale for flash photolysis?

A

1 milliseconds

77
Q

What can flash photolysis also be harnessed for specifically?

A

Can cause dissociation of CO from haemoglobin and myoglobin

78
Q

How is dissociation of CO from haemoglobin and myoglobin measured?

A

Can be measured optically on a timescale of 10^-15 s (femtoseconds)

79
Q

How can re-association of CO to haemoglobin and myoglobin measured?

A

On a 10^-12s timescale (picoseconds)

80
Q

How is the dissociation and re-association of CO onto haem/myoglobin observed?

A
  • Observed by optical techniques
  • When CO2 binds to haem you get a change in the ion oxidation state which gives a colour change
  • You can therefore use absorbance to measure this
81
Q

What are some examples of indirect quenching methods?

A
  1. Quenched flow
  2. Radioactive labelling
  3. HPLC (High performance liquid chromatography)
82
Q

In radioactive labelling, what changes and what remains the same?

A

The position of the radioactive label will alter in the reaction however the overall radioactivity will not change as the reaction proceeds

83
Q

What is necessary in a radioactive labelling reaction?

A

Quenching methods are necessary in order to stop the reaction, a second method necessary to separate the different constituents and a third to count the radioactivity in each

84
Q

How is radioactivity theoretically expressed?

A

In curies (Ci) or in becquerels (Bq)

85
Q

What do becquerels (Bq) refer to?

A

The rate of decomposition of the radioactive compound

86
Q

What can detect radioactivity?

A

A scintillation counter detects radioactivity in counts per minute (cpm)

87
Q

How does a scintillation counter work?

A

A chemical called a scintillant is added to the sample to be analysed and converts the radiation energy of the radioactive species into light quanta which can be measured using a standard photomultiplier in a machine called scintillation counter

88
Q

Using a scintillation counter, what amounts of compounds can be detected?

A

Depending on the radioactive atom, amounts of compound down to 10^-17M can be detected

89
Q

What are some examples of radioactive atoms?

A
  • 3H (tritium)
  • 14C
  • 35S
  • 32P
  • Atoms are available commercially
90
Q

What does HPLC stand for?

A

High performance liquid chromatography

91
Q

How is separation optimised in HPLC?

A

Using stronger column matrices, higher pressure and automated injection, in-line detection using the most sensitive optical methods such as absorbance and fluorescence

92
Q

Where is HPLC useful?

A

It is very useful in an assay where the reactants and products have an optical signal but cannot be differentiated until they have been physically separated

93
Q

What is used in HPLC when reactants and products do not have optical signals?

A

Refractive index detectors are widely used to detect and quantify different constituents

94
Q

How is the observed experimental signal recorded in HPLC?

A

As a function of the elution time or a volume from the HPLC column

95
Q

What does the area of each peak describe in a chromatogram?

A

A measure of the amount of each constituent in the injected sample

96
Q

What is not always clear in a chromatogram?

A
  • Which peak represents which chemical
  • This an be overcome as the chemical giving rise to a peak in the chromatogram can be further analysed using identification techniques such as mass spectrometry
97
Q

Where is mass spectrometry attached to on a HPLC machine?

A

The extra mass spectrometry instrument is often directly attached downstream of a HPLC machine

Biochemistry 2 (5 decks)

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