John C. Croney

Fluorescence: basic concepts, practical aspects, and some anecdotes.

We hope that we have conveyed information of interest and value to present and future fluorescence practitioners. Those readers with a sustaining interest in this topic may wish to consult more comprehensive sources such as Molecular Fluorescence: Principles and Applications, an excellent text by Valeur, or Principles of Fluorescence Spectroscopy by Lakowicz. Many specialized fluorescence topics are covered in the series Topics in Fluorescence Spectroscopy (Volumes 1-6), and several volumes of Methods in Enzymology (e.g., Volumes 246 and 278) have dealt with issues in fluorescence spectroscopy. Proceedings from the International Conference on Methods and Applications of Fluorescence Spectroscopy, 1997 (MAFS 97) and MAFS 98 (in press) also present fluorescence work on many different topics in biological and chemical fields. The Molecular Probes Handbook and web site (www.probes.com) are also rich sources of useful information. Finally, any reader with a question or seeking advice on some topic related to fluorescence is welcome to e-mail D.M.J. at djameson@hawaii.edu.

Fluorescence Polarization: Past, Present and Future

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.

Fluorescence spectroscopy in biochemistry: teaching basic principles with visual demonstrations

Although most biochemistry curricula include some treatment of light absorption and spectrophotometry, discussion of fluorescence spectroscopy is generally omitted. This omission is unfortunate given the increasing use of fluorescence in many fields of biochemical research. In this paper we briefly review the principles and applications of fluorescence in biochemical systems, from the viewpoint of teaching fluorescence in undergraduate curricula. Simple practical demonstrations are presented, which clearly demonstrate important concepts yet require minimal specialized equipment.

Conformational dynamics and temperature dependence of photoinduced electron transfer within self-assembled coproporphyrin:cytochrome c complexes.

The focus of the present study is to better understand the complex factors influencing intermolecular electron transfer (ET) in biological molecules using a model system involving free-base coproporphyrin (COP) complexed with horse heart cytochrome c (Cc). Coproporphyrin exhibits bathochromic shifts in both the Soret and visible absorption bands in the presence of Cc and an absorption difference titration reveals a 1:1 complex with an association constant of 2.63 +/- 0.05 x 10(5) M(-1). At 20 degrees C, analysis of time-resolved fluorescence data reveals two lifetime components consisting of a discrete lifetime at 15.0 ns (free COP) and a Gaussian distribution of lifetimes centered at 2.8 ns (representing (1)COP --> Cc ET). Temperature-dependent, time-resolved fluorescence data demonstrate a shift in singlet lifetime as well as changes in the distribution width (associated with the complex). By fitting these data to semiclassical Marcus theory, the reorganizational energy (lambda) of the singlet state electron transfer was calculated to be 0.89 eV, consistent with values for other porphyrin/Cc intermolecular ET reactions. Using nanosecond transient absorption spectroscopy the temperature dependences of the forward and thermal back ET originating from triplet state were examined ((3)COP --> Cc ET). Fits of the temperature dependence of the rate constants to semiclassical Marcus theory gave lambda of 0.39 eV and 0.11 eV for the forward and back triplet ET, respectively (k(f) = (7.6 +/- 0.3) x 10(6) s(-1), k(b) = (2.4 +/- 0.3) x 10(5) s(-1)). The differing values of lambda for the forward and back triplet ET demonstrate that these ET reactions do not occur within a static complex. Comparing these results with previous studies of the uroporphyrin:Cc and tetrakis (4-carboxyphenyl)porphyrin:Cc complexes suggests that side-chain flexibility gives rise to the conformational distributions in the (1)COP --> Cc ET whereas differences in overall porphyrin charge regulates gating of the back ET reaction (reduced Cc --> COP(+)).

Discrete Bathochromic Shifts Exhibited by Fluorescein Ligand Bound to Rabbit Polyclonal Anti-Fluorescein Fab Fragments

Eleven individual hyperimmune rabbit polyclonal anti-fluorescein Fab fragment preparations were resolved into heterogeneous subfractions based on differential dissociation times from a specific adsorbent. Four Fab subfractions (i.e., 0.1-, 1.0-, 10-, and 100-day elutions) that differed in affinity were characterized and classified according to the extent of the bathochromic shift in the absorption properties of antibody-bound fluorescein ligand. Absorption maxima of bound fluorescein were shifted in all cases to two distinct narrow ranges, namely, 505 to 507 nm or 518 to 520 nm relative to 491 nm for free fluorescein. There was no direct correlation between the two spectral shift populations and antibody affinity, fluorescence polarization, fluorescence quenching, or fluorescence lifetimes of bound ligand. Fluorescence emission maxima varied with the bathochromic shift range. Bound fluorescein ligand, with absorption maxima of 505 to 507 nm and 518 to 520 nm showed fluorescence emission maxima of 519 to 520 nm and 535 nm, respectively. The two spectral shift ranges differed by ∼14 to 15 nm and/or energies of ∼1.5 kcal mol−1 relative to each other and to the absorption maximum for free fluorescein. Spectral effects on the antibody-bound ligand were discussed relative to solvent-water studies and the atomic structure of a high-affinity liganded anti-fluorescein active site.

Fluorescence resonance energy transfer studies on anthrax lethal toxin

Anthrax lethal toxin is a binary bacterial toxin consisting of two proteins, protective antigen (PA) and lethal factor (LF), that self‐assemble on receptor‐bearing eukaryotic cells to form toxic, non‐covalent complexes. PA63, a proteolytically activated form of PA, spontaneously oligomerizes to form ring‐shaped heptamers that bind LF and translocate it into the cell. Site‐directed mutagenesis was used to substitute cysteine for each of three residues (N209, E614 and E733) at various levels on the lateral face of the PA63 heptamer and for one residue (E126) on LFN, the 30 kDa N‐terminal PA binding domain of LF. Cysteine residues in PA were labeled with IAEDANS and that in LFN was labeled with Alexa 488 maleimide. The mutagenesis and labeling did not significantly affect function. Time‐resolved fluorescence methods were used to study fluorescence resonance energy transfer (FRET) between the AEDANS and Alexa 488 probes after the complex assembled in solution. The results clearly indicate energy transfer between AEDANS labeled at residue N209C on PA and the Alexa 488‐labeled LFN, whereas transfer from residue E614C on PA was slight, and none was observed from residue E733C. These results support a model in which LFN binds near the top of the ring‐shaped (PA63)7 heptamer.

Resolution of rabbit polyclonal anti-fluorescein Fab (IgG) fragments into subpopulations differing in affinity and spectral properties of bound ligand.

Fab fragments derived from ten different IgG populations of hyperimmune rabbit polyclonal anti-fluorescein antibodies were further resolved into subfractions based on differences in time-dependent dissociation from an FITC-adsorbent in the presence of 0.1 M fluorescein at 4 degrees C. Fab fragments separated into subpopulations based on specific dissociation times of 0.1 day, 1.0 day, 10 days and 100 days from the adsorbent. Finally, after the 100 days elution step incubation with 6.0 M guanidine-HCl was included to determine total protein concentration of specific anti-fluorescein Fab fragments. Yields of specifically eluted Fab fragments ranged from 12.7 to 84.1% of the total Fab population originally incubated with the adsorbent. All Fab polyclonal populations and subpopulations analyzed quenched the fluorescence of the bound ligand by 90% or greater. None of the plots of protein concentration versus percent yield of the total specific antibody obtained for each of the five resolved fractions constituting a specific polyclonal population conformed to Gaussian distributions. All resolved Fab subpopulations retained bound fluorescein ligand that exhibited significant bathochromic shifts in absorbancy. Based on the extent of the red-shift the antibodies segregated into one of two general spectral families showing either a peak shift to 505-507 nm or to 518-520 nm. The red-shift to 518-520 nm appeared unique to rabbit anti-fluorescein antibodies, since corresponding large shifts have not been observed with antibodies derived from other species (e.g. mouse, rat, chicken, etc.). K(d) values determined for the resolved fractions confirmed a continuous progression in affinity from the 0.1day through the 100 days elution. Preliminary isoelectric focusing analyses revealed progressive selection for relatively more homogeneous fractions, especially in the 100 days resolved fraction.