Radioiodination of transforming growth factor-beta (TGF-beta) in a modified Bolton-Hunter reaction system.
We have optimized the use of the Bolton-Hunter reagent to prepare 125I-labeled transforming growth factor-beta (TGF-beta). Conditions were developed to obtain monovalent modification of human-recombinant TGF-beta 2 (hrTGF-beta 2) at a basic pH necessary for efficient protein acylation (> or = 26% of theoretical) while obviating the problems of TGF-beta aggregation/precipitation. The purified Bolton-Hunter labeled hrTGF-beta 2 had a specific activity of 1.8-2.1 microCi/pmol, and the 125I label was fully acid-precipitable. [125I]hrTGF-beta 2 was electrophoretically indistinguishable from unlabeled starting material and displayed full immunoreactivity with polyclonal anti-TGF-beta 2 antibody. Both hrTGF-beta 2 and Bolton-Hunter-labeled [125I]hrTGF-beta 2 inhibited the growth of mink lung epithelial cells with equal efficacy. These data validate a modified conjugation-iodination method for TGF-beta and invite general use of the Bolton-Hunter reagent for iodination of other TGF-beta isoforms and peptides similarly susceptible to precipitation/aggregation under standard Bolton-Hunter incubation conditions.
Ross
J
J
Research Department, Ciba-Geigy Corporation, Summit, NJ 07901.
Janero
D R
DR
Hreniuk
D
D
Wennogle
L P
LP
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D007553:Isotope Labeling / Q000379:methods*; D008168:Lung / Q000166:cytology / Q000187:drug effects; D008907:Mink; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000494:pharmacology; D013388:Succinimides; D016212:Transforming Growth Factor beta / Q000138:chemical synthesis* / Q000494:pharmacology
D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D013388:Succinimides; D016212:Transforming Growth Factor beta; C022581:Bolton-Hunter reagent