Azido ruthenium: a new photoreactive probe for calcium-binding proteins.
Ca2+, involved in almost all processes of cell life, mediates its activity through reversible interaction with specific binding sites in proteins. Although several Ca2+-dependent activities are known, many of the proteins responsible remain unidentified. Here we describe the synthesis, purification, characterization and potential uses of a new Ca2+-like reagent, azido ruthenium (AzRu), which can be photoactivated. AzRu strongly inhibits Ca2+-dependent activities. AzRu can be used to probe proteins in solution or embedded in membranes. AzRu has no effect on Ca2+-independent or Mg2+-dependent activity. After exposure to ultraviolet irradiation, AzRu binds covalently and specifically to Ca2+-binding proteins, thus providing a new approach for identifying and purifying Ca2+-binding proteins, for characterizing their Ca2+-binding sites and for exploring previously unknown Ca2+-dependent processes. In this protocol we also include a description of the preparation of [103Ru]AzRu, which can be used for labeling Ca2+-binding sites in proteins and identifying previously unknown Ca2+-binding proteins. The preparation of AzRu takes approximately 2-3 days.
Israelson
Adrian
A
Department of Life Sciences, The Zlotowski Center for Neuroscience and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
Zilberberg
Noam
N
Shoshan-Barmatz
Varda
V
D001386:Azides / Q000138:chemical synthesis* / Q000737:chemistry / Q000528:radiation effects; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000032:analysis* / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods; D007553:Isotope Labeling / Q000379:methods; D015336:Molecular Probe Techniques; D015335:Molecular Probes / Q000138:chemical synthesis* / Q000737:chemistry / Q000528:radiation effects; D009942:Organometallic Compounds / Q000138:chemical synthesis* / Q000737:chemistry / Q000528:radiation effects; D012429:Ruthenium Radioisotopes / Q000737:chemistry; D014466:Ultraviolet Rays
D001386:Azides; D002135:Calcium-Binding Proteins; D015335:Molecular Probes; D009942:Organometallic Compounds; D012429:Ruthenium Radioisotopes; C508266:azido ruthenium; D002118:Calcium