In Vitro Radiolabeling of Peptides and Proteins

Ton N.M. Schumacher1, Theodore J. Tsomides1

1 Massachusetts Institute of Technology, Cambridge
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 3.3
DOI:  10.1002/0471140864.ps0303s00
Online Posting Date:  May, 2001
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Abstract

Radiolabeling of peptides or proteins is often performed to enhance the sensitivity of detection, to quantitate the binding of peptides to other molecules, or for radioimmunoassays. This unit presents a variety of assays for radiolabeling peptides and proteins with 125I, 131I, 14C, and 3H.

     
 
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Table of Contents

  • Basic Protocol 1: Iodination at Tyrosine or Histidine Residues Using Iodo‐Beads
  • Alternate Protocol 1: Iodination at Tyrosine or Histidine Residues Using Chloramine T or Iodogen
  • Alternate Protocol 2: Iodination at Tyrosine or Histidine Residues Using Lactoperoxidase
  • Support Protocol 1: Equimolar Iodination to Give High Yields of Product
  • Support Protocol 2: Separation of Unlabeled Peptide from Iodinated Products by HPLC
  • Basic Protocol 2: Iodination at Lysine Residues or N‐Terminus Using Bolton‐Hunter Reagent
  • Basic Protocol 3: 14C or 3H Labeling at Lysine Residues or N‐Terminus by Acetylation Using Anhydride
  • Alternate Protocol 3: 14C or 3H Labeling at Lysine Residues or N‐Terminus by Reductive Alkylation
  • Alternate Protocol 4: 14C or 3H Labeling at Cysteine Residues Using Iodoacetic Acid or Iodoacetamide
  • Basic Protocol 4: 3H Labeling at Tyrosine Residues by Reduction of 127I‐Labeled Peptide
  • Basic Protocol 5: Introduction of Labeled Amino Acid Residues During Peptide Synthesis
  • Alternate Protocol 5: Selective Labeling on N‐Terminus During Peptide Synthesis
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Iodination at Tyrosine or Histidine Residues Using Iodo‐Beads

  Materials
  • Iodo‐Beads (Pierce)
  • 0.1 M sodium phosphate, pH 6.0 or pH 8.5
  • Carrier‐free sodium iodide‐125 (Na125I; Amersham, Du Pont NEN, or ICN Biomedicals) or sodium iodide‐131 (Na131I)
  • 0.1 M sodium hydroxide
  • Peptide, ideally dissolved in ≤0.4 ml of water or 0.1 M sodium phosphate
  • Methanol
  • HPLC solvent A: 0.1% (v/v) trifluoroacetic acid (TFA) in water
  • HPLC solvent B: 0.1% (v/v) TFA in acetonitrile
  • Filter paper
  • 100‐µl syringe with beveled tip, or hot pipettor (i.e., dedicated to radioactive use)
  • Luer‐tipped syringes (1‐, 5‐, and 20‐ml) and one 23‐G needle
  • Solid‐phase extraction device (e.g., Sep‐Pak or Sep‐Pak Plus C18 cartridge, Waters)
  • Polypropylene or other plastic tubes (12 × 75 mm)

Alternate Protocol 1: Iodination at Tyrosine or Histidine Residues Using Chloramine T or Iodogen

  • Anion‐exchange resin, e.g., Dowex 1‐X8, chloride form, 100 to 200 mesh (Bio‐Rad)
  • BSA solution: 1.0 mg/ml BSA in PBS, with 0.02% sodium azide (NaN 3)
  • 1.0 mg/ml chloramine T in 0.1 M sodium phosphate, prepared immediately before use
  • Iodogen (Pierce), dichloromethane, and glass tubes or vials (optional alternate to chloramine T)
  • Saturated solution of tyrosine in water (∼0.4 mg/ml at 25°C)
  • 2.5 mg/ml sodium metabisulfite (Na 2S 2O 5) in 0.1 M sodium phosphate, prepared immediately before use (optional alternate to tyrosine solution)
  • Pasteur pipet or 1‐ml syringe with glass wool plug inserted at bottom
  • Gel‐filtration column, e.g., Sephadex G‐10 or G‐25 (Pharmacia Biotech), or Excellulose Desalting Column (Pierce; optional alternate to anion‐exchange column)

Alternate Protocol 2: Iodination at Tyrosine or Histidine Residues Using Lactoperoxidase

  • 0.2 mg/ml lactoperoxidase in PBS
  • 0.01% hydrogen peroxide (H 2O 2) in PBS (diluted from 30% stock immediately before use)
  • 2 U/ml glucose oxidase in PBS and 0.1 M D‐glucose in PBS (optional alternate to H 2O 2)

Support Protocol 1: Equimolar Iodination to Give High Yields of Product

  Materials
  • 0.1 M sodium borate, pH 8.5
  • 0.1% (w/v) gelatin in 0.1 M sodium borate, pH 8.5
  • 125I‐labeled Bolton‐Hunter reagent (Amersham, Du Pont NEN, or ICN Biomedicals); or substitute unlabeled Bolton‐Hunter reagent and iodinate later
  • Peptide, ideally ≥1 mg/ml in ≤100 µl of 0.1 M sodium borate, pH 8.5
  • 1 M glycine in 0.1 M sodium borate, pH 8.5
  • Gel‐filtration column, e.g., Sephadex G‐10
  • Dry nitrogen tank and outlet tubing fitted with needle
  • Polypropylene or other plastic tubes (12 × 75 mm)

Support Protocol 2: Separation of Unlabeled Peptide from Iodinated Products by HPLC

  Materials
  • Saturated sodium acetate solution
  • Peptide, ideally ≥1 mg/ml in ≤100 µl H 2O or sodium acetate
  • [14C]‐ or [3H]acetic anhydride (Amersham, Du Pont NEN, ICN Biomedicals, or Sigma)
  • Acetic anhydride, unlabeled (optional)

Basic Protocol 2: Iodination at Lysine Residues or N‐Terminus Using Bolton‐Hunter Reagent

  Materials
  • 0.2 M sodium borate, pH 9.0
  • Peptide, ideally ≥1 mg/ml in 0.1 to 1.0 ml of 0.2 M sodium borate, pH 9.0
  • [3H]sodium borohydride (Amersham, Du Pont NEN, or ICN Biomedicals) or [3H]sodium cyanoborohydride (Amersham)
  • 0.01 M NaOH
  • 37% (12.4 M) formaldehyde stock
  • [14C]formaldehyde and unlabeled sodium cyanoborohydride (optional alternative for labeling with 14C instead of 3H)

Basic Protocol 3: 14C or 3H Labeling at Lysine Residues or N‐Terminus by Acetylation Using Anhydride

  Materials
  • 0.5 M Tris⋅Cl/6 M guanidine⋅HCl/0.002 M disodium EDTA, pH 8.6
  • Peptide, ideally ≥1 mg/ml in 0.1 to 1.0 ml 0.5 M Tris⋅Cl/6 M guanidine⋅HCl/0.002 M disodium EDTA, pH 8.6
  • Dithiothreitol
  • 1.0 M NaOH
  • [14C]‐ or [3H]iodoacetic acid or [14C]iodoacetamide (Amersham, Du Pont NEN, or ICN Biomedicals)
  • Iodoacetic acid or iodoacetamide, unlabeled (optional)

Alternate Protocol 3: 14C or 3H Labeling at Lysine Residues or N‐Terminus by Reductive Alkylation

  Materials
  • L‐Leucine, unlabeled
  • L[14C]‐ or L‐[3H]leucine, 1 to 5 mCi
  • Triethylamine
  • 2‐(t‐butoxycarbonyloxyimino)‐2‐phenylacetonitrile (BOC‐ON, Aldrich)
  • Dioxane
  • Diethyl ether, anhydrous
  • 1.0 M HCl, 4°C
  • Separatory funnel
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Figures

Videos

Literature Cited

   Bolton, A.E. and Hunter, W.M. 1973. The labelling of proteins to high specific radioactivities by conjugation to a 125I‐containing acylating agent. Biochem. J. 133:529‐539.
   Chersi, A., Trinca, M.L., and Camera, M. 1988. 14C‐labeling of synthetic peptides. J. Immunol. Methods 110:271‐273.
   Crestfield, A.M., Moore, S., and Stein, W.H. 1963. The preparation and enzymatic hydrolysis of reduced and S‐carboxymethylated proteins. J. Biol. Chem. 238:622‐627.
   Edelhoch, H. 1962. The properties of thyroglobulin. VIII. The iodination of thyroglobulin. J. Biol. Chem. 237:2778‐2787.
   Eisen, H.N. and Keston, A.S. 1949. The immunologic reactivity of bovine serum albumin labeled with trace amounts of radioactive iodine (131I). J. Immunol. 63:71‐80.
   Fraenkel‐Conrat, H. and Colloms, M. 1967. Reactivity of tobacco mosaic virus and its protein toward acetic anhydride. Biochemistry 6:2740‐2745.
   Fraker, P.J. and Speck, J.C., Jr. 1978. Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6‐tetrachloro‐3α,6α‐diphenylglycoluril. Biochem. Biophys. Res. Commun. 80:849‐857.
   Gahmberg, C.G. and Andersson, L.C. 1977. Selective radioactive labeling of cell surface sialoglycoproteins by periodate‐tritiated borohydride. J. Biol. Chem. 252:5888‐5894.
   Greenwood, F.C., Hunter, W.M., and Glover, J.S. 1963. The preparation of 131I‐labelled human growth hormone of high specific radioactivity. Biochem. J. 89:114‐123.
   Hubbard, A.L. and Cohn, Z.A. 1975. Externally disposed plasma membrane proteins. I. Enzymatic iodination of mouse L cells. J. Cell Biol. 64:438‐460.
   Hunter, W.M. and Greenwood, F.C. 1962. Preparation of iodine‐131 labelled human growth hormone of high specific activity. Nature 194:495‐496.
   Jentoft, N. and Dearborn, D.G. 1983. Protein labeling by reductive alkylation. Methods Enzymol. 91:570‐579.
   Marchalonis, J.J. 1969. An enzymatic method for the trace iodination of immunoglobulins and other proteins. Biochem. J. 113:299‐305.
   Marchalonis, J.J., Cone, R.E., and Santer, V. 1971. Enzymic iodination: A probe for accessible surface proteins of normal and neoplastic lymphocytes. Biochem. J. 124:921‐927.
   Markwell, M.A.K. 1982. A new solid‐state reagent to iodinate proteins: Conditions for the efficient labeling of antiserum. Anal. Biochem. 125:427‐432.
   Rizzino, A. and Kazakoff, P. 1991. Iodination of peptide growth factors: Platelet‐derived growth factor and fibroblast growth factor. Methods Enzymol. 198:467‐479.
   Sarin, V.K., Kent, S.B.H., Tam, J.P., and Merrifield, R.B. 1981. Quantitative monitoring of solid‐phase peptide synthesis by the ninhydrin reaction. Anal. Biochem. 117:147‐157.
   Stewart, J.M. and Young, J.D. 1984. Solid Phase Peptide Synthesis. Pierce Chemical Co. Rockville, Ill.
   Tack, B.F. and Wilder, R.L. 1981. Tritiation of proteins to high specific activity: Application to radioimmunoassay. Methods Enzymol. 73:138‐147.
   Thean, E.T. 1990. Comparison of specific radioactivities of human α‐lactalbumin iodinated by three different methods. Anal. Biochem. 188:330‐334.
   Tsomides, T.J. and Eisen, H.N. 1993. Stoichiometric labeling of peptides by iodination on tyrosyl or histidyl residues. Anal. Biochem. 210:129‐135.
   Wood, F.T., Wu, M.M., and Gerhart, J.C. 1975. The radioactive labeling of proteins with an iodinated amidination reagent. Anal. Biochem. 69:339‐349.
   Wolff, J. and Covelli, I. 1969. Factors in the iodination of histidine in proteins. Eur. J. Biochem. 9:371‐377.
Key References
   Means, G.E. and Feeney, R.E. 1971. Chemical Modification of Proteins. Holden‐Day, Inc., San Francisco, Calif.
  Excellent overview of side chain chemistries, including but not limited to radiolabeling procedures.
   Tsomides and Eisen 1993. See above.
  Describes use of Sep‐Pak to remove free iodine, HPLC to separate iodinated products, Edman degradation to identify products, and pH dependence of tyrosine vs. histidine iodination using Iodo‐Beads.
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