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Radioacttve labeled pepttdes and proteins are used extensively in many areas of biochemtstry, pharmacology, and medicme Forexample, they are frequently employed as tracer molecules in quantitative determmattons, such as measurement of hormone andhormone-receptor concentrations, and kinetic and equihbrmm studies of both agonist and antagomst bmding to receptors Thesestudies require the accurate determmation of very low amounts of the labeled peptides and protem Such small amounts can beaccurately measured by using a tracer molecule labeled to high-specific radloactivity. The most commonly used radlonuchdesfor pepttde and protem studies are trttmm and 125I, followed by t4C, 35S and 32P.
In most cases where [ 125I]todme is used to label a molecule, It is a foretgn label, i.e, it does not normally occur in the molecule The replacementof nonradioactive carbon or hydrogen by [14C]carbon or tritmm will have virtually no effect on the biologtcal properties of the molecule. However, the replacement ofa proton with a large iodme atom can have a considerable effect on the properties of the protein, this can usually be overcomeIf the label is at a position that is some distance from the site of biological activity
There are several major advantages in using [125I]todme over [ 14C]carbon or trmum The first is the specific activity available (Table 1)
There is an mverse relationshtp between the half hfe of an isotope and its theoretical specific activity. In some Isotopesthis maximum is never obtamable. [125I] Iodme has a maxtmum theoretical specific activity of 2 175 Cl/mm01 and is usually obtainable at -2000 Wmmol. The maxtmumspecific acttvmes of [14C]carbon and trmum are 62 4 mWmmo1 and 28.8 Wrnmol, respectively. Several atoms of [14C]carbon or trmum can be substituted in a molecule, but the specific activity obtamed is still very much lower than with [251]iodme Very small amounts of radiotodmated material can be used while mamtaming sensitive assays. The count rate obtainedfrom [125I]iodme can be 100 times greater than for trmum and 35,000 times greater than [i4C]carbon Another major advantage is in thecase of detection [125I]iodme decays by electron capture followed by X-ray emission which can be counted directly in a y counter [125I]iodme is used in viva for imaging owing to the nonparticulate emission which reduces radiation damage to the biologicalmaterial. Both [ 14C]carbon and trltlum are pure p-emitters resulting in particulate emlsslon in the form of electrons. To count these, scmtlllantsand a scmtlllatlon counter are required, which mvolves extra sample preparation and counting time, extra cost of scmtlllantand increased volumes of radioactive material for disposal The high speclfic actlvlty and count rate of lodmated compoundsare advantageous in autoradlography, especially when very small amounts of receptor are to be localized In contrast, the timerequired to autograph trltlated and [14C]carbon hgands can stretch to months
Table 1 Half-Life and Available Specific Activity of Commonly Used Isotopes
Radionuclide | Type of emission | Half-life | Specific activity (per millimole) at 100% isotopic abundance |
---|---|---|---|
125I | γ(EC) | 60 00 d | 2175 0 C1 |
131I | γ and β- | 8 04 d | 16,235 0 C1 |
14C | β- | 5730 00 yr | 62 4 mC1 |
3H | β- | 12 43 yr | 28 8Q |
32P | β- | 14 30 d | 6000 0 C1 |
35S | β- | 87 40 d | 1493 0 C1 |
Complex organic chemistry may be required to label a molecule with [ 14C]carbon This can mean starting from [14C]-labeled COZ, methanol, BaC03, benzene, and so forth It is also an expensive radlonuchde to obtain, and multistage preparationsmevltably decrease overall yields. [ 14C]-peptldes (unl