Lanthanides proteins

G. Site-specific labeling of proteins with a rigid lanthanide-binding tag. ChemEioChem 2006, 7,1599-1504. 

Homogeneous Time Resolved Fluorescence (HTRF) (Cisbio International) is an assay based on the proximity of a lanthanide cryptate donor and a fluorescent acceptor molecule whose excitation wavelength overlaps that of the cryptate s emission. The utility of this technique is based on the time resolved fluorescence properties of lanthanides. Lanthanides are unique in the increased lifetime of their fluorescence decay relative to other atoms, so a delay in collection of the emission intensity removes the background from other fluorescent molecules. An example of the HTRF assay is a generic protein-protein interaction assay shown in Fig. 2. 

An alternative approach is that adopted by Horrocks and co-workers, where the aromatic residues in metal-binding proteins are used as sensitizers. Since the distance between the metal and the donor is effectively fixed, this provides a rigid scaffold for the experiment, and the absence of a directly conjugated pathway between the metals means that Forster (through space) energy transfer can be assumed. The r-6 distance-dependence of this means that the extent of sensitized emission from the lanthanide ion provides information on the spatial relationship between the metal-ion binding site (lanthanide ions often bind at Ca2+ sites) and nearby aromatic residues. 58-60... 

The DELFIA assay, the first effective lanthanide-based immunoassay, was developed and commercialized by the early 1980s.108-112 DELFIA (Dissociation Enhanced Lanthanide Fluoro-ImmunoAssay) is a heterogeneous assay which uses a lanthanide complex based on aminocarboxylate ligands such as EDTA, EGTA, or DTPA, linked to the antibody by reaction of appended isothiocyanate groups (e.g., complex (45)) with nucleophilic residues, particularly amines, on the protein surface (Figure 11). 

The overall distribution of lanthanides in bone may be influenced by the reactions between trivalent cations and bone surfaces. Bone surfaces accumulate many poorly utilized or excreted cations present in the circulation. The mechanisms of accumulation in bone may include reactions with bone mineral such as adsorption, ion exchange, and ionic bond formation (Neuman and Neuman, 1958) as well as the formation of complexes with proteins or other organic bone constituents (Taylor, 1972). The uptake of lanthanides and actinides by bone mineral appears to be independent of the ionic radius. Taylor et al. (1971) have shown that the in vitro uptakes on powdered bone ash of 241Am(III) (ionic radius 0.98 A) and of 239Pu(IV) (ionic radius 0.90 A) were 0.97 0.016 and 0.98 0.007, respectively. In vitro experiments by Foreman (1962) suggested that Pu(IV) accumulated on powdered bone or bone ash by adsorption, a relatively nonspecific reaction. On the other hand, reactions with organic bone constituents appear to depend on ionic radius. The complexes of the smaller Pu(IV) ion and any of the organic bone constituents tested thus far were more stable (as determined by gel filtration) than the complexes with Am(III) or Cm(III) (Taylor, 1972). 

It is likely that identification of lanthanide(III)-protein complexes would be even more difficult than for the actinide-protein complexes because of their lower stability and greater tendency to dissociate during separation processes. 

Even though the specific protein that binds the lanthanides may or may not be transferrin, the behavior of high-specific activity lanthanides (< 1 jtM/kg7) compared with that of lanthanides administered with significant amounts of stable carrier strongly suggests the kind of break-through one would expect if a protein transport system were overloaded. As long as the amount of lanthanide introduced into the... 

Figure 9.51 Time-resolved FRET assay systems involve energy transfer between the lanthanide chelate and an organic dye that are brought together as two labeled molecules bind to an analyte. In this illustration, an antibody labeled with a lanthanide chelate is used along with a Cy5-labeled antibody to detect a protein target in solution. Excitation of the lanthanide label results in energy transfer and excitation of the cyanine dye only if they are held within close enough proximity to allow efficient FRET to occur. Under these conditions, excitation of the lanthanide chelate results in cyanine dye emission, which will not occur if the labeled antibodies have not bound to a target.

Hemmila, 1988) (see Chapter 9, Section 9). The most commonly used lanthanides for this purpose are europium (Eu3+), terbium (Tb3+), and samarium (Sm3+). Proteins modified with DTTA and complexed with lanthanide metal ions form the basis for unique fluorescent probes possessing long lived signals upon excitation. 

Figure 16.7 The ECAT mass tag consists of a DOTA metal chelate group that can coordinate a lanthanide metal ion and a bromoacetyl group for coupling to cysteine-containing proteins.

The use of mass tagging reagents to analyze proteomic data has greatly improved the ability to compare samples for protein expression differences. However, a major limitation of the ICAT procedure (Section 1, this chapter) is that it can only compare two samples simultaneously, usually a test and a control. Even with the ECAT design (Section 2) using multiple lanthanide metals to make a series of different mass tag signatures, it is difficult to extend the... 

Franz, K.J., Nitz, M., and Imperiali, B. (2003) Lanthanide-binding tags as versatile protein coexpression probes. Chem. Bio. Chem. 4, 265-271. 

Sculimbrene, B.R., and Imperiali, B. (2006) Lanthanide-binding tags as luminescent probes for studying protein interactions./. Am. Chem. Soc. 128(22), 7346-7352. 

GFP hopo ICBP IP3 Ln3+ mal memal MLCK nota oxine par pdta pmea py quin-2 green fluorescent protein hydroxypyridinon(at)e intestinal calcium-binding protein inositol 1,4,5-triphosphate a lanthanide(III) cation malonate methylmalonate myosin light chain kinase 1,4,7-triazacyclononane-l,4,7-triacetate 8- hydroxyquinoline pyridine-2-azo-4 -dimethylaniline propylene-1,2-diaminetetraacetate 9- [2-(phosphonomethoxy)ethyl] adenine pjrridine pjrridyl 8-amino-2- [(2-amino-5-methylphenoxy )methyl] -6-methoxyquinoline-ATJV -tetraacetate 2- [ [2-[his(carboxymethyl)amino]-5-methyl-phenoxy] methyl] -6-methoxy-8- [bis(carboxymethyl) amino] quinoline]... 

---