Iodination of annexin V for imaging apoptosis.
Our goal in this investigation was to develop a method for iodinating annexin V that would be suitable for the in vivo detection of apoptosis.


METHODS
Annexin V was iodinated with (125)I using 2 different techniques: direct iodination with IODO-BEADS, resulting in the iodination of tyrosine residues; and use of the Bolton-Hunter reagent, which binds to lysine. The active fraction of the labeled preparation was purified by affinity chromatography. We assessed thyroid accumulation of free iodide by comparing mice with blocked and unblocked thyroids. We tested the ability of iodinated annexin V to bind apoptotic cells in vitro using irradiated neuroblastoma cells and immobilized phosphatidylserine and in vivo using C3H mice subjected to whole-body irradiation.


RESULTS
The efficiency of IODO-BEADS iodination was just below 30%; with the Bolton-Hunter protocol we were able to achieve 40% efficiency. When the IODO-BEADS-labeled preparation was injected into nude mice, activity accumulated rapidly in the thyroid. Two hours after injection, uptake in the thyroid region was clearly visible on a gamma-camera scan. This uptake was absent in mice that had had their thyroids blocked. We concluded that the IODO-BEADS method of labeling resulted in a protein that was rapidly deiodinated in vivo. By contrast, when annexin V was labeled using the Bolton-Hunter protocol, there was no evidence of activity accumulating in the thyroid. The Bolton-Hunter-labeled annexin V bound to apoptotic cells and immobilized phosphatidylserine in vitro. The active fraction of Bolton-Hunter-labeled annexin V was approximately 0.75. In C3H mice given 5-Gy whole-body irradiation, there was a significant induction of apoptosis in the spleen, as measured by the terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay, and a 4-fold increase in (125)I activity in the spleens relative to that of the control animals.


CONCLUSIONS
Direct iodination of annexin V on tyrosine residues is a poor technique suffering from rapid deiodination in vivo. With Bolton-Hunter chemistry, one can produce a molecule that retains its label in vivo and binds to apoptotic cells in vitro and in vivo.
Russell
James
J
Department of Medical Physics, Memorial Sloan Kettering Cancer Center, New York, New York 10021, USA. russellj@mskcc.org
O'Donoghue
Joseph A
JA
Finn
Ron
R
Koziorowski
Jacek
J
Ruan
Shutian
S
Humm
John L
JL
Ling
C Clifton
CC
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism*; D017209:Apoptosis; D006801:Humans; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D051379:Mice; D008809:Mice, Inbred C3H; D013388:Succinimides
D017304:Annexin A5; D007457:Iodine Radioisotopes; D013388:Succinimides; C022581:Bolton-Hunter reagent