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Radioactivity resides in UNSTABLE
NUCLEUS.
Nucleus decays with EMISSION OF RADIATION.
Many elements have both:
stable isotopes (non-radioactive, eg
12C),
and
unstable isotopes (radioactive, eg
14C).
Different numbers of neutrons in nucleus.
Electronic configuration the same as that of non-radioactive
isotope of same element, so chemical properties are the same.
Hence use in chemistry and biology of
RADIOACTIVE TRACERS
Substitute radioactive for stable isotope, undergoes exactly same
reactions but can be detected and measured as required by radiation
monitoring device.
Types of Radioactive Decay
a-emission
Nucleus disintegrates with emission of alpha particles (ionized He nuclei, He2+).
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eg
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226
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Ra --->
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222
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Rn +
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4
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He
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88
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86
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2
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Only for elements of high atomic no. (>80), little used in biochemistry.
b-emission
Nucleus disintegrates with emission of electron (b- particle), or less commonly a positron (b+ particle).
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eg
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14
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C --->
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14
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N + b-
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6
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7
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g-emission
Disintegrating nucleus emits radiation (hn),
high energy, ionising cf
X-rays.
Gamma ray emission can occur along with emission of beta particles, or as a
result of other processes eg electron
capture, or sometimes without affecting atomic no. of nucleus.
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eg
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125
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I + e- (capture) --->
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125
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Te + g
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53
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52
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Detection and Measurement of Radioactivity
Geiger-Muller counter
Radiation causes ionization of gas in tube
---> current flow.
Portable, useful for monitoring of spillages.
Scintillation Counters
Preferred for most quantitative work:
Radiation from radio-isotope ---> excitation of electrons in a SCINTILLANT
or FLUOR ---> emission of LUMINESCENCE, measure with photodetector.
- Solid scintillation
counter
(gamma counter)
g-rays emerge from sample tube -
impinge on external scintillant crystal
(NaI/T1I) --> emits light pulses to photomultiplier.
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Liquid scintillation counter
(beta counter)
b-particles often too weak to use
external fluor.
Sample mixed in solution with "scintillation cocktail". Captures b-emission at source ---> photons. May
be 2-stage process involving primary and secondary fluors.
Units of Radioactivity
Fundamental (and SI) unit is the Becquerel (Bq)
which is the number of DISINTEGRATIONS PER SECOND (dps),
ie. the number of nuclei
that break down per second.
For historical reasons, radioactivity often measured in Curie (Ci) units.
1Ci = 3.7 x 1010 Bq
Because of the magnitudes, common derived units are :
the microCurie
(mCi)
the megaBecquerel
(MBq)
Measuring device reads counts per minute (cpm).
In a scintillation counter each "count" = pulse of light from fluor activated by radiation.
Counting efficiency < 100%, because of:
- radiation escaping
without activating fluor
- fluors
undergoing quenching
- hn from fluors
not reaching photodetector
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Bq =
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cpm
60
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x
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100
counting efficiency (%)
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Specific Radioactivity
This is radioactivity per gram or per mole of compound
Isotopically labelled
compounds usually diluted with an excess of unlabelled compound (carrier)
in order to:
- use biologically
relevant concentrations without excessive radiation hazard
- avoid
excessive loss of isotope by adsorption etc.
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Specific activity increases as{
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labelled species
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} increases
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carrier
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Isotope dilution analysis depends on principle of adding labelled species of known specific activity then
measuring specific activity of a recovered sample, hence calculate amount of
unlabelled species in sample.
Decay Kinetics: Half-Life
Disintegrations of radioactive nuclei in sample are in proportion to
number present (1st order kinetics), so isotope decays exponentially. (Holme & Peck Ch 5).
Half-life (t0.5)= Time for no. of radioactive nuclei to decay
by half
Important factor in planning experiments with isotopes.....
Long t0.5 (eg 14C, 5570
years):
- no complications due
to loss of isotope over duration of experiment, but
- significant hazard if
ingested (long-term exposure)
Shorter t0.5 (eg 32P, 14.2
days)
- plan purchase so
delivery only when ready to use
- allow for decay during
experiment (especially if measuring, eg
metabolic elimination)
Biochemical Applications of Isotopes
Tracers
To observe metabolic fates of species. eg
mechanism of photosynthesis:
CO2 + H2O ---> (CH2O) + O2
Reaction carried out using 18O labelled
CO2 ---> No 18O recovered as O2. Some in
H2O.
If using 18O labelled H2O,
all 18O recovered in O2.
So better representation of overall process is:
CO2 + 2H2O ---> (CH2O) + O2 +
H2O {ie CO2
+ 2H2O ---> (CH2O) + O2 + H2O}
Enzyme assay
CH3CO.~SCoA + *CO2 -----> -OO*C-CH2CO.~SCoA
acetyl -CoA
malonyl-CoA
Measure fixation of *C (14C). (Becomes non-volatile)
Isotope Dilution Analysis
Radioautography (Autoradiography)
Detect position of specific labelled species on
chromatogram or electrophoretogram
Into which protein has labelled glycine gone?
or
Which DNA fragment has bound labelled probe?
Place on top of photographic film. Incubate in light proof (& radiation
proof) container. Develop. Radiation produces an image.
Largely supplanted by luminescent probes.
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