Radiolabeling of Peptides and Proteins
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Radioacttve labeled pepttdes and proteins are used extensively in many areas of biochemtstry, pharmacology, and medicme For example, they are frequently employed as tracer molecules in quantitative determmattons, such as measurement of hormone and hormone-receptor concentrations, and kinetic and equihbrmm studies of both agonist and antagomst bmding to receptors These studies require the accurate determmation of very low amounts of the labeled peptides and protem Such small amounts can be accurately measured by using a tracer molecule labeled to high-specific radloactivity. The most commonly used radlonuchdes for pepttde and protem studies are trttmm and 125 I, followed by t4C, 35S and 32P.
In most cases where [ 125 I]todme is used to label a molecule, It is a foretgn label, i.e, it does not normally occur in the molecule The replacement of nonradioactive carbon or hydrogen by [14 C]carbon or tritmm will have virtually no effect on the biologtcal properties of the molecule. However, the replacement of a proton with a large iodme atom can have a considerable effect on the properties of the protein, this can usually be overcome If the label is at a position that is some distance from the site of biological activity
There are several major advantages in using [125 I]todme over [ 14 C]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 Isotopes this maximum is never obtamable. [125 I]Iodme has a maxtmum theoretical specific activity of 2 175 Cl/mm01 and is usually obtainable at -2000 Wmmol. The maxtmum specific acttvmes of [14 C]carbon and trmum are 62 4 mWmmo1 and 28.8 Wrnmol, respectively. Several atoms of [14 C]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 obtained from [125 I]iodme can be 100 times greater than for trmum and 35,000 times greater than [i4C]carbon Another major advantage is in the case of detection [125 I]iodme decays by electron capture followed by X-ray emission which can be counted directly in a y counter [125 I]iodme is used in viva for imaging owing to the nonparticulate emission which reduces radiation damage to the biological material. Both [ 14 C]carbon and trltlum are pure p-emitters resulting in particulate emlsslon in the form of electrons. To count these, scmtlllants and a scmtlllatlon counter are required, which mvolves extra sample preparation and counting time, extra cost of scmtlllant and increased volumes of radioactive material for disposal The high speclfic actlvlty and count rate of lodmated compounds are advantageous in autoradlography, especially when very small amounts of receptor are to be localized In contrast, the time required to autograph trltlated and [14 C]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 |
|---|---|---|---|
|
125 I |
γ(EC ) |
60 00 d |
2175 0 C1 |
|
131 I |
γ and β- |
8 04 d |
16,235 0 C1 |
|
14 C |
β- |
5730 00 yr |
62 4 mC1 |
|
3 H |
β- |
12 43 yr |
28 8Q |
|
32 P |
β- |
14 30 d |
6000 0 C1 |
|
35 S |
β- |
87 40 d |
1493 0 C1 |
Complex organic chemistry may be required to label a molecule with [ 14 C]carbon This can mean starting from [14 C]-labeled COZ, methanol, BaC03, benzene, and so forth It is also an expensive radlonuchde to obtain, and multistage preparations mevltably decrease overall yields. [ 14 C]-peptldes (unless using reductive methylatlon) must be built from labeled ammo acids. Trltlum labeling requires synthesis of specific precursors Trltlatlon of samples often involves catalytic hydrogenation to add to a double bond or to replace a halogen in a molecule. Radlolodmatlons are comparatively easy
There are also disadvantages in using lodme As previously stated, lodme is usually a foreign label and labeling with [125 I]lodme can therefore alter the properties of many molecules This can be a particular problem in receptor studies Even a small change in structure, such as oxldatlon of one ammo acid in an lodmatlon, can completely block bmding to the receptor Reaction rates can also be altered The advantage of having a high specific activity is countered by the disadvantage of a shorter half-life There is also the posslblhty of faster decomposltlon, especially radiation decomposltlon and also the loss of iodine. A shorter half-life is advantageous for waste disposal [ 14 C]Labeled materials can remam pure for many years
