New azulene derivatives containing resonantly electron withdrawing formyl and resonantly electron donating fluorine substituents on the seven-as well as the five-member ring have been prepared. Systematic color changes as a result of perturbation of HOMO, LUMO, and LUMO + 1 energies were discussed. Corresponding excited-state properties related to S 2 -fluorescence were determined.

Activation of the proenzyme form of the malarial protease PfSUB-1 involves the autocatalytic cleavage of an Asp-Asn bond within the internal sequence motif (215)LVSADNIDIS(224). A synthetic decapeptide based on this sequence but with the N- and C-terminal residues replaced by cysteines (Ac-CVSADNIDIC-OH) was labeled with 5- or 6-isomers of iodoacetamidotetramethylrhodamine (IATR). The doubly labeled peptides have low fluorescence because of ground-state, noncovalent dimerization of the rhodamines. Cleavage of either peptide by recombinant PfSUB-1 results in dissociation of the rhodamine dimers, which abolishes the self-quenching and consequently leads to an approximately 30-fold increase in the fluorescence. This spectroscopic signal provides a continuous assay of proteolysis, enabling quantitative kinetic measurements to be made, and has also enabled the development of a fluorescence-based assay suitable for use in high-throughput screens for inhibitors of PfSUB-1. The structure of the rhodamine dimer in the 6-IATR-labeled peptide was shown by NMR to be a face-to-face stacking of the xanthene rings. Time-resolved fluorescence measurements suggest that the doubly labeled peptides exist in an equilibrium consisting of rhodamines involved in dimers (closed forms) and rhodamines not involved in dimers (open forms). These data also indicate that the rhodamine dimers fluoresce and that the associated lifetimes are subnanosecond.

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Fluorescence polarization was first observed in 1920 and during the next few decades the theoretical foundations of the phenomenon were clearly established. In the last two decades of the 20(th) century, fluorescence polarization became one of the most prevalent methods used in clinical and biomedical sciences. In this article we review the history of fluorescence polarization, its theoretical foundations and some of the more important practical developments, which helped to popularize the method. We also discuss important, but often misunderstood, practical considerations including the wavelength dependence of the limiting polarization and the effect of energy transfer on polarization. The present state of fluorescence polarization, both in pure research as well as in the applied biosciences is also reviewed. Finally, we speculate on possible future developments in the field, such as the use of multi-photon techniques.

In this report the temperature dependence of photoinduced electron transfer between both singlet- and triplet-state uroporphyrin (1URO and 3URO, respectively) and the heme group of ferricytochrome c (Cc) has been examined. Analysis of the forward electron-transfer rate originating from 1URO as a function of temperature gives a reorganizational energy (λ) of 1.0 ± 0.02 eV with an associated electronic coupling term (H AB) of (3.7 ± 0.3) × 10-4 eV. In addition, the singlet lifetime data exhibit a temperature-dependent Gaussian distribution that narrows as the temperature increases. This suggests a conformational distribution of bound porphyrin with a narrow range of potential wells at lower temperatures. The corresponding temperature dependence of the electron transfer between 3URO/URO+ and the heme group of Cc gives a λ = 0.49 ± 0.05 eV/0.19 ± 0.03 eV and H AB ∼ 4 × 10-4 eV/1.4 × 10-4 eV, respectively. These observations are consistent with orientational dependent conformational states as well as possible gating of electron transfer within the complex.