SYNTHESIS OF A HIGHLY LUMINESCENT TERBIUM CHELATE AND ITS APPLICATION TO ACTIN

We synthesized a highly luminescent terbium chelate which consists of DTPA as a chelating group, phalloin as an actin-binding ligand and an aromatic ring as a sensitizer of Th emission. The synthesis was based on the property of DTPA dianhydride that has the capacity to react with two amines, while retaining its ability to chelate Tb3+ (the resulting DTPA possesses three carboxyl groups). We screened 17 kinds of aromatic compounds for their ability to sensitize Th emission with near UV excitation. All of the compounds tested had both a 6-membered ring and one or two amino group(s). Among them, cytosine was the best sensitizer. From the peak-peak ratio, the fluorescence intensity of a solution of Tb-DTPa-cytosine-phalloin-labeled actin was about one-fourth of an equimolar solution of tetramethylrhodamine. The excitation peak was about 302 nm. Emission from a single muscle fiber stained with this new Th chelate was so intense that we could accumulate sufficient photon counts in a short time. Also, actin bundles stained with the Th chelate were able to be visualized under a fluorescence microscope. The stability constant of the chelator was 10(22.46) M-1 for Tb3+. This value was very similar to that of DTPA, which possessed five carboxyl groups. The new Tb chelate had additional expediencies. It was electrically neutral, which made it quite suitable for studying electrostatic circumstances around the terbium site on actin. We present data on this issue. Tb3+ itself is optically isotropic. However, cytosine of the Th chelate has a polarized absorption transition dipole moment. Therefore, in conjunction with the long-lived excited state, the synthesized Tb chelate has potential of serving to probe the slow rotational relaxation of actin filaments. We can simply replace phalloin with other agents to label not only actin but also various proteins. Such Th chelates will be a quite useful tool for a variety of biological studies.