David M. Jameson

[12] Fluorescence anisotropy applied to biomolecular interactions

Publisher Summary This chapter focuses on the applications of polarization or anisotropy data. The appeal of fluorescence spectroscopy in the study of biomolecular systems lies in the characteristic time scale of the emission process, the sensitivity of the technique, and its ability to accommodate rapid and facile changes in the solvent milieu under conditions corresponding to thermodynamic equilibrium. On excitation of a fluorescent solution and observation of the emission at right angles both to the direction of propagation of the exciting light and to the direction of the electric vector, the polarization of the emission is defined in this chapter. The optical densities of the solution may be unavoidably high (even allowing for smaller cuvette or front-face geometries), and the requisite inner filter corrections may lower confidence in the binding data. Polarization/anisotropy measurements—such as lifetime measurements—are intensive quantities and as such are not subject to first-order inner filter corrections.

Tryptophan emission from human hemoglobin and its isolated subunits.

Abstract— The emission spectra of human adult hemoglobin A0 and its isolated α and ß subunits were obtained using a highly sensitive photon‐counting spectrofluorometer. The quantum yields of the emissions, relative to free tryptophan, were also measured as well as the excitation polarization spectra for hemoglobin A0 and apohemoglobin. The fluorophore bis‐ANS was utilized to probe for the presence of apoproteins in the hemoprotein preparations. The work suggests that tryptophan may be useful as an intrinsic probe to study dynamical processes in hemoglobin.

A multifrequency phase fluorometer using the harmonic content of a mode-locked laser

ABSTRACT We describe the construction and operation of a cross-correlation phase and modulation fluorometer which uses the harmonic content of a high repetition rate mode-locked laser as the excitation source. A mode-locked argon ion laser is used to synchronously pump a dye laser. The pulse train output from the dye laser is amplitude modulated by an acousto-optic modulator and then frequency doubled with an angle tuned frequency doubler. With the particular dye utilized in these studies, the ultraviolet light obtained was continuously tunable over the range 280-310 nm. In the frequency domain the high repetition rate pulsed source gives a large series of equally spaced harmonic frequencies. The frequency spacing of the harmonics is determined by the repetition frequency of the laser. Amplitude modulation of the pulse train permits variation of the frequency quasi-continuously from a few hertz to gigahertz. Use of cross-correlation techniques permits precise isolation of individual frequencies. The cross...

Multifrequency cross‐correlation phase fluorometer using synchrotron radiation

The construction and operation of a cross‐correlation phase and modulation fluorometer using the synchrotron radiation facility at the ADONE–Frascati electron storage ring is described. In the frequency domain the high repetition rate pulsed source gives a large series of equally spaced harmonic frequencies. Use of cross‐correlation techniques in conjunction with such a light source permits one to isolate one harmonic frequency from the adjacent frequencies with high precision. The cross‐correlation frequency required for the analysis of the phase delay and modulation ratio is obtained using two coupled frequency synthesizers, one of which drives the radio‐frequency cavity of the storage ring and the other which modulates the response of the photomultipliers used for the signal detection. The accuracy, reproducibility, and sensitivity of the instrumentation have been determined on a number of systems and are reported.

Spectroscopic and molecular modeling studies of caffeine complexes with DNA intercalators

Recent studies have demonstrated that caffeine can act as an antimutagen and inhibit the cytoxic and/or cytostatic effects of some DNA intercalating agents. It has been suggested that this inhibitory effect may be due to complexation of the DNA intercalator with caffeine. In this study we employ optical absorption, fluorescence, and molecular modeling techniques to probe specific interactions between caffeine and various DNA intercalators. Optical absorption and steady-state fluorescence data demonstrate complexation between caffeine and the planar DNA intercalator acridine orange. The association constant of this complex is determined to be 258.4 +/- 5.1 M-1. In contrast, solutions containing caffeine and the nonplanar DNA intercalator ethidium bromide show optical shifts and steady-state fluorescence spectra indicative of a weaker complex with an association constant of 84.5 +/- 3.5 M-1. Time-resolved fluorescence data indicate that complex formation between caffeine and acridine orange or ethidium bromide results in singlet-state lifetime increases consistent with the observed increase in the steady-state fluorescence yield. In addition, dynamic polarization data indicate that these complexes form with a 1:1 stoichiometry. Molecular modeling studies are also included to examine structural factors that may influence complexation.

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.

Effect of docosahexaenoic acid on membrane fluidity and function in intact cultured Y-79 retinoblastoma cells

Considerable metabolic energy is expended in ensuring that membranes possess a characteristic fatty acid composition. The nature of the specific requirement of the retina for high levels of docosahexaenoic acid (DHA) is as yet undefined. Previous work has speculated that DHA is required to maintain the fluid nature and permeability necessary for optimal retinal function. Cultured Y-79 retinoblastoma cells were grown in serum-containing media with and without supplemental DHA. Resultant changes in membrane fluidity were assessed using fluorescent probes. No differences were observed in rotational probe mobility as assessed by fluorescence polarization despite a fourfold increase in cellular DHA content. Lateral probe mobility as assessed by pyrene eximer formation was significantly enhanced in DHA-supplemented cells. Both the DHA content and total fatty acid unsaturation index in retinoblastoma cells were directly correlated with membrane fluidity as reported by eximer formation (Pearsons rho = 0.96 and 0.92, respectively). DHA supplementation also resulted in a significant increase in cellular choline uptake. We speculate that the effect of DHA content on retinal function may be mediated by changes in membrane fluidity and associated enzyme and transport activities.

Fluorescence polarization: measurements with a photon-counting photometer.

A T-format, photon-counting polarization photometer was constructed to facilitate precise polarization measurements at low signal-to-noise ratios. The instrument optics and electronics are described. Several examples of the instruments performance are given, including measurements on picomolar fluorescein solutions at low excitation resolution and the excitation polarization spectrum of indole (2 x 10(-4)M) at a resolution of 5 A.

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.

Time-resolved fluorescence studies on site-directed mutants of human serum albumin.

Human serum albumin (HSA) contains a single tryptophan residue at position 214. The emission properties of tryptophan 214 from recombinant albumins, namely, normal HSA, FDH‐HSA and a methionine 218 HSA were examined. In all cases, the excited state lifetimes were best described by a two component model consisting mainly of a Lorentzian distribution. The centers of these distributions were 5.60 ns for HSA, 4.23 ns for FDH‐HSA, and 6.08 ns for Met‐218 HSA. The global rotational correlation times of the three HSAs were near 41 ns while the amplitude and rate of the local motion varied. These changes in the lifetimes and mobilities suggest perturbation in the local protein environment near tryptophan 214 as a consequence of the amino acid substitutions.