Direct binding probes

Molecular imaging focuses on the visualization of molecules and molecular processes. Thus, especially in the case of direct-binding probes, the imaging tech-... 

There are two general types of probes commonly used in PET imaging direct binding probes and indirect probes. In the former, the labelled species directly binds... 

Bogyo, M., Shin, S., McMastee, J. S., and Ploegh, H. L. Substrate binding and sequence preference of the proteasome revealed by active-site-directed affinity probes. Chem Biol 1998, 5, 307-320. 

Amine-terminated, full-generation PAMAM and PPI dendrimers, as well as carboxylate-terminated half-generation PAMAM dendrimers, can directly bind metal ions to their surfaces via coordination to the amine or acid functionality. A partial hst of metal ions that have been bound to these dendrimers in this way includes Na+, K+, Cs+, Rb+, Fe +, Fe +, Gd +, Cu+, Cu +, Ag+, Mn +, Pd, Zn, Co, Rh+,Ru +,andPt + [18,19,27,36,54,82-96]. Tuxro et al.have also shown that the metal ion complexes, such as tris(2,2 -bipyridine)ruthenium (Rulbpylj), can be attached to PAMAM dendrimer surfaces by electrostatic attraction [97]. A wide variety of other famihes of dendrimers have also been prepared that bind metal ions to their periphery. These have recently been reviewed [3]. Such surface-bound metal ions can be used to probe dendrimer structure using optical spectroscopy, mass spectrometry, and electron paramagnetic resonance (EPR) [86-88,90,97-99]. 

The a-chloroacetamide group has features that are beneficial for undirected ABPP. Its small size does not bias binding elements towards a specific class of enzyme, and it possesses reactivity towards a broad variety of nucleophilic amino acid residues. A library of a-chloroacetamide-based probes were synthesized by Cravatt s group. The binding element in these probes was a dipeptide that was varied with small, large, hydrophobic, and charged side chains, and a biotin or rhodamine tag was appended as a reporter tag. Upon screening of eukaryotic proteomes with this library, many enzymes previously unaddressed by directed ABPP probes were uncovered. These included fatty acid synthase, hydro-xypyruvate reductase, malic enzyme, and the nitrilase superfamily [163, 164]. In contrast to the sulfonate esters, a-chloroacetamides react preferentially with cysteine residues in the proteome. 

The use of Ty fluorescence quenching as a probe provides a direct spectroscopic probe of fluoride binding to Tymet. Stopped-flow fluorimetric experiments can be performed with high sensitivity and speed, thereby avoiding the need for large amounts of protein and the experimental difficulties arising from the intrinsic instability of the enzyme, the latter being important especially at the lower pH values. 

Direct binding of fluo- [44e - (i)] rescently labeled tool probe against GST-tagged purified proteins (9000 proteins available)... 

As the result of high specificity and sensitivity, nucleic acid probes are in direct competition with immunoassay for the analytes of some types of clinical analytes, such as infectious disease testing. Assays are being developed, however, that combine both probe and immunoassay technology. In such hybrid probe—immunoassays, the immunoassay portion detects and amplifies the specific binding of the probe to a nucleic acid. Either the probe per se or probe labeled with a specific compound is detected by the antibody, which in turn is labeled with an enzyme or fluorophore that serves as the basis for detection. 

Although FeMo-cofactor is clearly knpHcated in substrate reduction cataly2ed by the Mo-nitrogenase, efforts to reduce substrates using the isolated FeMo-cofactor have been mosdy equivocal. Thus the FeMo-cofactor s polypeptide environment must play a critical role in substrate binding and reduction. Also, the different spectroscopic features of protein-bound vs isolated FeMo-cofactor clearly indicate a role for the polypeptide in electronically fine-tuning the substrate-reduction site. Site-directed amino acid substitution studies have been used to probe the possible effects of FeMo-cofactor s polypeptide environment on substrate reduction (163—169). Catalytic and spectroscopic consequences of such substitutions should provide information concerning the specific functions of individual amino acids located within the FeMo-cofactor environment (95,122,149). 

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