Radiolabeling of Peptides and Proteins

  • Arvind C. Patel
  • Stewart R. Matthewson
Part of the Springer Protocols Handbooks book series (SPH)


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 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 replacement of nonradioactive carbon or hydrogen by [14C]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 [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 Isotopes this 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 maxtmum specific 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 obtained from [125I]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 [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 biological material. Both [ 14C]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 [14C]carbon hgands can stretch to months
Table 1

Half-Life and Available Specific Activity of Commonly Used Isotopes


Type of emission


Specific activity (per millimole) at 100% isotopic abundance

125 I


60 00 d

2175 0 C1

131 I

γ and β-

8 04 d

16,235 0 C1

14 C


5730 00 yr

62 4 mC1

3 H


12 43 yr

28 8Q

32 P


14 30 d

6000 0 C1

35 S


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 preparations mevltably decrease overall yields. [ 14C]-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 [125I]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 [ 14C]Labeled materials can remam pure for many years


Label Protein Hypochlorous Acid Sodium Metabisulfite Tracer Molecule Comprehensive Guide 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Houghten, R A and Ll, C H (1979) Reduction of sulfoxldes in peptldes and proteins Anal Bzochem 98,36–46CrossRefGoogle Scholar
  2. 2.
    Hunter, W in and Greenwood, F C (1962) Preparation of iodine 13 1-labelled growth hormone of high specific activity Nature 194,495–496PubMedCrossRefGoogle Scholar
  3. 3.
    Marchalonrs, J J (1969) An enzymlc method for the trace lodmatlon of mununoglobulms and other proteins Bzochem J 113,299–305Google Scholar
  4. 4.
    Huber, R E, Edwards, L A, and Carne, T J (1989) Studies on the mechanism of the todmatlon of tyrosme by lactoperoxtdase J Bzol Chem 264(3), 1381–1388Google Scholar
  5. 5.
    Murphy, M J (1976) 125I labelling of erythropolmon wrthout loss off of biologtcal Blochem J 159,287–289Google Scholar
  6. 6.
    Karonen, S L, Morsby, P, Stren M, and Seuderlug, U (1975) An enzymattc solid phase method for trace todmatton of proteins and pepttdes with 125I0dme Anal Bzochem 67, 1–10CrossRefGoogle Scholar
  7. 7.
    Fraker, P J and Speck, J C (1978) Protem and cell membrane todmatlons with sparingly soluble chloramtde, 1,3,4,6-tetrachloro-3a,6a-dtphenylglycolur11 BBRC 80(4), 849–857Google Scholar
  8. 8.
    Markwell, M A K (1982) A new sobd-state reagent to lodmate proteins Anal Blochem 125,427–432CrossRefGoogle Scholar
  9. 9.
    Bolton, A E and Hunter, W in (1973) The labelling of proteins to high specific radtoactlvmes by coqugatton to 125I-contaming acylating agent Bzochem J 133, 529–539Google Scholar
  10. 10.
    Tang, Y S, et al (1982) N-succmtmtdyl proptenate characterisatlon and optimum condtttons for use as a trttmm labelling agent for proteins Lab Comp Radlopharm 20,277–284Google Scholar
  11. 11.
    Dottavto-Martin, D and Ravel, J in (1978)Radlolabeling of proteins by reductive alkylatlon with carbon-14 formaldehyde and sodmm cyanoborohydrtde Anal Bzochem 87,562–565CrossRefGoogle Scholar
  12. 12.
    Guide to Radlozodznatzon Techwques(1993) Amersham International plc publication,Little Chalfont, UKGoogle Scholar

Copyright information

© Humana Press Inc , Totowa, NJ. 1998

Authors and Affiliations

  • Arvind C. Patel
    • 1
  • Stewart R. Matthewson
    • 1
  1. 1.Amersham InternationalAmershamUK

Personalised recommendations