Fluorescence binding titration

Figure 7.36 Peptide association with GroEL (a) Fluorescence emission spectra from fluorescence binding titration experiment with fixed concentration of AMPH+ peptide(0.5 / M) titrated with GroEL until saturation in buffer pH 7.5 at 20°C. Note blue shift and increase in emission intensity as titration progresses. (b) Three binding isotherms from three fluorescence binding titration experiments involving the indicated peptides under conditions described in (a) (Reproduced from Preuss et al., 1999, Fig. 6).

Many of the fully aromatic 6-6-5 tricyclics show good fluorescence properties, and in some instances this has been utilized in biochemical applications. The reader is directed to the literature cited in Section 7.22.12.1 (benzo-separated purines) for examples of how the sensitivity of the fluorescence properties of tricyclic fluorophores to environmental conditions can be utilized to analyze the binding parameters associated with biomacromolecules. Of particular interest in this regard is a method known as fluorescence-polarization titration (83B2347). 

Brandt, U., and von Jagow, G., 1991, Analysis of inhibitor binding to the mitochondrial cytochrome c reductase by fluorescence quench titration. Evidence for a icatalytic switch at the Qo center, Eur. J. Biochem. 195 163nl70. 

Murakami and Berliner (1983) later reported the existence of a zinc binding site in bovine, human, guinea pig, and rabbit a-lactalbumins, in which the zinc site is physically distinct from the site for binding calcium. This proposal was supported by the fact that when a cation binds to one site, the ensuing conformational shift excludes binding to the other site. All metal ions that were bound to apo-a-iactalbumin at the calcium site caused the same fluorescence shift. Titration of Ca(II) or Mn(II) protein with Zn(II) or Al(III) caused a complete return to apo-a-lactalbumin fluorescence parameters. In contrast, titration of apo-a-iactalbumin with Zn(II) caused no change in fluorescence parameters. 

Figure S.1S. Fluorescence intensity titration curve. Fluorescence intensity decreases wMi recombinant, ftjll-lergth Xenopus XPA concentration increases due to XPA binding to the single fluorescein labeled at the 5 -end of a ds 50-mer oligonucleotide. The titral on curve is saturated (levels off) at 100 nM XPA Cutlosey from William R. Wiley tnvironmental Molecular Sciences Laboratories.

Solutions of e-ADP were titrated with the helicase (Fig ure 8.33). In the absence of rylamide, there was little change in the e-ADP fluorescence. The titrations were performed again, in solutions with increasing amounts of acrylamide. Under these conditions, e-ADP showed an increase in fluorescence upon binding. This increase occurred because the e-ADP became shielded from acrylamide upon binding to helicase. The authors also showed that acrylamide had no effect on the affinity of e-ADP for helicase. In this system the use of a quencher allowed measurement of a binding reaction that would otherwise be difficult to measure. 

Fluorescence DNA binding titrations are performed in the reverse mode by adding 5.0 jjlI aliquots of a 1.0 mM DNA stock to 3.0 ml of 5 pM protein in a 4 ml fluorescence quartz cell (Fig. 6). A carefully calibrated Unimetrics syringe with a utility stop permits reproducible, repetitive deliveries of a constant volume. The solution is mixed by constant stirring using a magnetic flea at the bottom of the cell. Nucleic acid and protein stock concentrations are determined by UV... 

The 1 2 binding constants for a ditopic biscrown ether chiral oligo(/ -phenylenevinylene) host (COPY) toward sodium (Na+) was determined by a fluorescence spectroscopy titration (Figure 9) in chloroform. ... 

Analysis of 1 2 binding equilibria by calorimetric titration is based on (51), (64), (66), and (68) in the same fashion as illustrated above in the case of UV-vis and fluorescence spectroscopy titration, except that we use the enthalpy changes for the 1 1 (A//hg) and 1 2 (A/fHG2) complexes and need to add the total volume (V) to these equations. Thus, for calorimetric titrations (after correcting for dilution effects as mentioned in Section 2.2.4), we obtain (81), (82), (83), and (84). 

The highest reported binding constant to date for a mono-macrocyclic calixarene or calixarene analogue receptor and Cgo is 3.48 x 10 which was ascribed to the Ag(I) A-heterocyclic carbene-bridged calixarene 48 synthesized by Quin et al. [72]. This binding constant for the 1 1 complexation was determined by fluorescence quenching titration study of in acetonitrile at 298 K (Fig. 33.33). 

---