Stoichiometry fluorescence-labeled

The stoichiometry of the recharged DNA/PLL/SPLL particles was studied using sucrose-gradient ultracentrifugation of fluorescently labeled polyion complexes in 25 mM HEPES buffer. Rhodamine-labeled DNA (Rh-DNA) and either fluorescein-labeled PLL (Fl-PLL) or SPLL (Fl-SPLL) were used to determine their relative amounts within DNA... 

BODIPY 530/550 C3 is insoluble in aqueous solution, but it may be dissolved in DMF or DMSO as a concentrated stock solution prior to addition of a small aliquot to a reaction. Coupling to amine-containing molecules may be done using the EDC/sulfo-NHS reaction as discussed in Chapter 3, Section 1.2 (Figure 9.29). However, modification of proteins with this fluorophore probably won t yield satisfactory results, since BODIPY fluorophores are easily quenched if substitutions on a molecule exceed a 1 1 stoichiometry. For labeling molecules which contain only one amine group, such as DNA probes modified at the 5 end to contain an amine (Chapter 27, Section 2.1), BODIPY 530/550 C3 will give intensely fluorescent derivatives. 

After first verifying that bead-bound monomers had minimal affinity for target RNA 12, the entire 11,325-compound library was screened with fluorescently-labeled HIV-1 FSS RNA as a pool. Fluorescent beads were then removed, cleaved, and cleaved material analyzed. As the stoichiometry of the library screen was set up such that bead-bound monomers were in excess (a choice made so as to favor dimmer formation on bead rather than in solution), mass spectrometry revealed only the identity of component monomers rather than the full structure of selected compounds. Nevertheless, this provided a substantial simplification of the library in a single step three monomers were identified as being selected in replicate experiments, potentially representing six unique compounds, or 0.05% of the library (ignoring terminus differentiation due to resin attachment taking this into consideration raises the number of compounds to a still-low 9). 

NCD-4 is a nonfluorescent carbodiimide derivative that forms a fluorescent adduct with the Ca -ATPase, accompanied by inhibition of ATPase activity and phos-phoenzyme formation [376-378]. Ca protected the enzyme against the inhibition by NCD-4 and reduced the extent of labeling, suggesting that the reaction may involve the Ca " " binding site. The stoichiometry of the Ca -protected labeling was i 2mole/mol ATPase. The fluorescence emission of the modified Ca -ATPase is consistent with the formation of a protein bound A-acylurea adduct in a relatively hydrophobic environment. After tryptic proteolysis of the NCD-4 labeled ATPase the fluorescence was associated with the A2 band of 24 kDa [376,379]. 

Since BODIPY fluorophores are easily quenched if substitutions on a molecule exceed a 1 1 stoichiometry, modification of proteins with this fluorophore probably will not yield satisfactory results. However, for labeling molecules that contain only one amine group, BODIPY FL C3-SE will give intensely fluorescent derivatives. 

Figure 16. LIF excitation (scale in nm) and emission (scale in cm ) spectra of VII with methanol and water [90a-c]. The electronic origins of the LIF excitation spectra of various complexes are labeled A (complex of stoichiometry 1 3), B (1 2), C (1 1) and D (1 1). The spectra of the complexes with water do not show the low-energy parts A and B. The fluorescence spectra were recorded with a spectral resolution of the detection monochromator of 10-15 nm (the position of excitation is indicated by an asterisk).

The interaction of selectively spin and dansyl mono-labeled neurotoxin II derivatives with solubilized nicotinic AChR protein isolated from Torpedo marmorata electric organ, was monitored by EPR and fluorescence spectroscopy(42). All the compounds specifically bind to the AChR, with dissociation constants ranging from 3 to 80 nM. The stoichiometry of the toxin-AChR complex is 2 1, assuming a molecular weight of 250 000 daltons for AChR(51). The two binding sites were found to be independent and indiscernible by dissociation constants and neurotoxin II spin label microenvironment, however they were not identical in that the nontoxic hexatrifluoro-acetyl neurotoxin II interacts only with one AChR binding site. 

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