Improved green fluorescence

This is an overview of some of the important, challenging, and problematic issues in contemporary electron transfer research. After a qualitative discussion of electron transfer, its time and distance scales, energy curves, and basic parabolic energy models are introduced to define the electron transfer process. Application of transition state theory leads to the standard Marcus formulation of electron transfer rate constants. Electron transfer in solution is coupled to solvent polarization effects, and relaxation processes can contribute to and even control electron transfer. The inverted region, in which electron transfer rate constants decrease with increasing exoergicity, is one of the most striking phenomena in electron transfer chemistry. It is predicted by both semiclassical and quantum mechanical models, with the latter appropriate if there are coupled high- or medium-frequency vibrations. The intramolecular reorganizational energy has different contributions from different vibrational modes, whic...

Contemporary Issues in Electron Transfer Research

The use of Forster or fluorescence resonance energy transfer (FRET) as a spectroscopic technique has been in practice for over 50 years. A search of ISI Web of Science with just the acronym "FRET" returns more than 2300 citations from various areas such as structural elucidation of biological molecules and their interactions, in vitro assays, in vivo monitoring in cellular research, nucleic acid analysis, signal transduction, light harvesting and metallic nanomaterials. The advent of new classes of fluorophores including nanocrystals, nanoparticles, polymers, and genetically encoded proteins, in conjunction with ever more sophisticated equipment, has been vital in this development. This review gives a critical overview of the major classes of fluorophore materials that may act as donor, acceptor, or both in a FRET configuration. We focus in particular on the benefits and limitations of these materials and their combinations, as well as the available methods of bioconjugation.

Materials for Fluorescence Resonance Energy Transfer Analysis: Beyond Traditional Donor-Acceptor Combinations

In the past there has been emphasis on the view that bioluminescent systems exhibit considerable diversity, both biologically and chemically [55]. While this certainly remains so, evidence is developing for interesting interand intra-phyletic similarities, especially in the structure of luciferins and the chemiluminescent reaction mechanisms. Bacteria. Although the reaction mechanism in bacteria is distinctly different from other well characterized systems, cross phyletic relationships between bacteria and other forms do occur. Luminous bacteria 'inhabit' other phyla; they are known to occur as symbionts in fish and other higher organisms [55), and thereby provide the host organism with a light source which may be used for one or more specific functions. The flashlight fish (Photohlepharon palpebratus), which possesses a pair of light organs situated just beneath the eyes, is a recently studied example [93]. (These fish are now on exhibit a t the Steinhart Aquarium in San Francisco.) Leiognathid fish, with internal luminous organs, have also been investigated [15,57, 581. The ability to luminesce occurs in several groups of marine bacteria, whose systematic relationships to each other and to nonluminous marine bacteria were described in detail by Reichelt and Baumann [ 105,1061. Luminous bacteria which are symbiotic fall within the genus Photobacteriurn; bacteria in the Beneckea genus have not been found in a symbiotic relationship and are generally isolated from seawater (K. Nealson and J. W. Hastings, unpublished). A more specific habitat for and function of the luminescence in the Beneckea species have not yet been uncovered. Insight concerning the function of the light emission is emerging from studies of the control of the synthesis of the luminescent system. In both Photobacterium and Beneckea species, luciferase is not synthesized constitutively [58, 951. A substance (termed autoinducer) synthesized by the cells themselves accumulates in the medium and results in the induction of luciferase synthesis [41]. By radioactive labeling, 5 peptides have been isolated which are specifically synthesized during the induction period [%I. From studies of mutants which require exogenous aldehyde for luminescence (see below), it was concluded that at least two genes are specifically involved in the in t.im biosynthesis of aldehyde factor for luminescence and that both genes are coordinately controlled with that for luciferase [29]. Luciferase synthesis is also subject to repression by glucose, reversible by cyclic

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1751-1097.1976.tb07282.x

Bioluminescence and chemiluminescence

Luciferases are enzymes that emit light in the presence of oxygen and a substrate (luciferin) and which have been used for real-time, low-light imaging of gene expression in cell cultures, individual cells, whole organisms, and transgenic organisms. Such luciferin-luciferase systems include, among others, the bacterial lux genes of terrestrial Photorhabdus luminescens and marine Vibrio harveyi bacteria, as well as eukaryotic luciferase luc and ruc genes from firefly species (Photinus) and the sea pansy (Renilla reniformis), respectively. In various vectors and in fusion constructs with other gene products such as green fluorescence protein (GFP; from the jellyfish Aequorea), luciferases have served as reporters in a number of promoter search and targeted gene expression experiments over the last two decades. Luciferase imaging has also been used to trace bacterial and viral infection in vivo and to visualize the proliferation of tumour cells in animal models.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/bio.676

Imaging of light emission from the expression of luciferases in living cells and organisms: a review.

Chemiluminescence in organic chemistry

Five esters of luciferin were synthesized and compared with native luciferin as substrates for firefly luciferase expressed in live intact mammalian cells. The esters themselves were not substrates for purified luciferase, but four were substrates for a purified esterase and all appeared to be hydrolysed to luciferin within mammalian cells. At a substrate concentration of 0.01 mM, the peak luminescence from the cos cells expressing luciferase was up to 6-fold greater with the esters than with unmodified luciferin. At 0.1 mM, the difference between luciferin and the esters was decreased. The kinetics of the luminescent signal with the different luciferin esters varied significantly, indicating possible differences in the rates of uptake, breakdown and enzyme inhibition. The esters did not support luminescence from Escherichia coli cells expressing firefly luciferase, suggesting a lack of appropriate esterase activity in this particular strain. The esters could be useful for the assay of luciferase expression in intact mammalian cells when luciferin levels are limiting, for example in tissues, and in plants. Alternative luciferin derivatives may allow further improvements in sensitivity.

/pdf/membrane-permeable-luciferin-esters-for-assay-of-firefly-3ppxxryqu6.pdf

Membrane-permeable luciferin esters for assay of firefly luciferase in live intact cells.

Isolable dioxetans usually afford triplet rather than singlet excited products but we now show that high yields of excited singlet states are obtainable from stable dioxetans by appropriate substitution; a remarkable rate acceleration of the chemiluminescent reaction by surfaces is observed.

Singlet excited states from dioxetan decomposition

It is suggested that dioxetans substituted by, or reacting with, compounds with strongly electron-donating groups decompose via radical ions and that excitation occurs by electron transfer.

Frank McCapra

Papers

Chemiluminescence in organic chemistry

Membrane-permeable luciferin esters for assay of firefly luciferase in live intact cells.

Singlet excited states from dioxetan decomposition

Alternative mechanism for dioxetan decomposition

Bacterial bioluminescence - identification of fatty acid as product, its quantum yield and a suggested mechanism