Covalent binding probes

Certain fluorescent compounds, such as formycin nucleotides and eosin, behave as noncovalently binding ATP analogs, and their fluorescence increases upon association with the ATPases. The transition from the Na+-form to the K+-form of Na+-K+-ATPase gives rise to a decrease in fluorescence from these probes. The fluorescence of fluorescein isothiocyanate (FITC) bound covalently at the nucleotide site of Na+-K+-ATPase (Table 3) also decreases in relation to transition from the Na+-form to the K+-form. With the noncovalent as well as with the covalently-binding probes the fluorescence decrease probably reflects the structural change in the nucleotide site associated with the reduced nucleotide affinity in the E2 form (see above). By contrast, when FITC is attached to the SR Ca2+-ATPase (at the residue homologous to the FITC-binding residue in Na+-K+-ATPase) the fluorescence increases upon removal of Ca2+. Most likely this relates to the above discussed difference between Na+-K+-ATPase and Ca2+-ATPase with respect to the existence of a stable E2 form with low affinity for nucleotide. 

Synthesis of Cannabinoid Receptor Ligands Approaches and Methods 3.1. Classical Cannabinoids and Covalent Binding Probes... 

Fig. 6.14. Quenched activity based probe for the imaging of cathepsins. Upon covalent binding of the sensor to histidine and serine residues in the active site of the enzyme, the quencher is released and increased fluorescence indicates the now covalently labeled enzyme of interest. 

Dunn SMJ, Blanchard SG, Raftery MA. 1980. Kinetics of carbamoylcholine binding to membrane-bound acetylcholine receptor monitored by fluorescence changes of a covalently bound probe. Biochemistry 19 5645-5652. 

Chitosan (Fig. 16) is an amino-modified natural polysaccharide that canbe also used as a polymeric gel for the covalent binding of OND probes [61]. Chitosan offers several advantages for NA immobilization. Its pH responsive properties allow it to be easily immobilized onto glass slides for the construction of arrays. Specifically, chitosan is soluble at low pH, when its amine groups are protonated, but becomes insoluble when the pH is raised above its pKa 6.3). 

Chemically modified DNAs can also be used as hybridization probes, provided that the modification does not interfere with the formation of hybrid DNA molecules. A psoralen biotin label has also been developed. Psoralen is a photoactivable agent that can intercalates into single- or double-stranded nucleic acids. On irradiation at 365 nm, it will covalently bind to the probes. This labeling reaction is simple and straightforward. However, the reagents for labeling and detection are only available in a kit format. 

Several useful schemes for attaching nucleic acid probes onto electrode surfaces have thus been developed [2-8]. The exact immobilization protocol often depends on the electrode material used for signal transduction. Common probe immobilization schemes include attachment of biotin-functionalized probes to avidin-coated surfaces [15], self-assembly of organized monolayers of thiol-functionalized probes onto gold transducers [16], carbodiimide covalent binding to an activated surface [17], as well as adsorptive accumulation onto carbon-paste or thick-film carbon electrodes [15-30]. 

In a similar biochemically oriented vein, several papers were published during 2003 in the journal Chemical Research in Toxicology, representing yet another area of applicability of 3 mm NMR probe capabilities. In the first of these reports, Hankin et al.159 described the results of an investigation into the covalent binding of leukotriene A4 to DNA and RNA. Later in 2003, Blair and co-workers160 reported on the characterization of 2 -deoxycytidine adducts derived from 4-oxo-2-nonenal, a novel lipid peroxidation product. Two of the adducts characterized, Ai and A2, were consistent with substituted ethano-deoxycytidine structures. The third adduct, B, was characterized as a 7-heptanone-etheno-deoxycytidine adduct (78). 

The immobilization of dsDNA and ssDNA to surfaces can be attained very easily by adsorption. No reagents or DNA modifications occur since the immobilization does not involve formation of covalent bonds with the surface. Surface immobilization of ssDNA by covalent binding, described by Thompson and co-workers [12], is convenient in DNA-hybridization sensing because it enables probe structure flexibility with respect to changes in its conformation to occur, such that hybridization can take place without the probe being removed from the sensor surface. However, non-selective adsorption of non-complementary ssDNA, added to bulk solution, also occurs at multiple sites in the interstitial regions on the sensor surface between immobilized ssDNA strands. The effects of selective and non-selective binding influence the detection of hybridization of the immobilized strands, as found by ICrull and co-workers [13]. 

Figure 20 Mechanism-based probe for cytochrome P-450 targeting. Oxidative activation of the probe by the enzyme generates a reactive ketene that covalently binds within the active site. 

---