Radioactive Isotopes

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⁺).

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).

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.

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.

   

• 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)

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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

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.

  Specific activity increases as{	labelled species	} increases
  	carrier

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 (t_0.5)= Time for no. of radioactive nuclei to decay by half

Important factor in planning experiments with isotopes.....
Long t_0.5 (eg ¹⁴C, 5570 years):

• no complications due to loss of isotope over duration of experiment, but
• significant hazard if ingested (long-term exposure)

Shorter t_0.5 (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 Aplications 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
See 13.4,
also cf stable isotope dilution analysis by mass spectrometry (5.4.1)

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.

Radioimmunoassay
The original form of competitive-binding immunoassay. Labelled (usually ¹²⁵I) and unlabelled antigen (Ag) compete for limited antibody (Ab).
Ag + Ab + Ag* <--> mixture of Ab-Ag & Ab-*Ag
Separate *Ag from Ab-*Ag (eg by precipitation with second antibody), and quantify Ab-*Ag.
As Ag in test sample is increased, radioactive Ab-*Ag decreases.
Calibrate for quantitative assay.