Optimal modification of annexin V with fluorescent dyes.
The many uses of chemically modified annexin Vs necessitate an understanding of the optimal degree of modification and modification sites of the protein. When reacted with the N-hydroxysuccinimide ester of Cy5.5, annexin V with one modification per mole of protein retained its affinity for phosphatidylserine of apoptotic cells, whereas modification with two dyes per mole of protein caused a complete loss of activity. A tryptic digest LC/MS method was used to identify the modification sites as either of two closely spaced lysine residues, in position 286 or 290. The crystal structure indicated the location of these lysines was distal to the phosphatidylserine binding sites on annexin V. These results can be used to develop active or inactive fluorescent control annexin V proteins and to suggest strategies for attaining higher levels of modification with retention of bioactivity.
Harvard Medical School, Center for Molecular Imaging Research, Charlestown, MA 02129, USA.
Eyk A
EA
Schellenberger
Ralph
R
Weissleder
Lee
L
Josephson
D017304:Annexin A5 / Q000737:chemistry*; D001665:Binding Sites; D002232:Carbocyanines / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes / Q000737:chemistry*; D008239:Lysine; D013058:Mass Spectrometry; D010718:Phosphatidylserines; D013388:Succinimides / Q000737:chemistry
D017304:Annexin A5; C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D005456:Fluorescent Dyes; D010718:Phosphatidylserines; D013388:Succinimides; D008239:Lysine; C001426:N-hydroxysuccinimide