Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay.

Small-molecule fluorescent probes embody an essential facet of chemical biology. Although numerous compounds are known, the ensemble of fluorescent probes is based on a modest collection of modular “core” dyes. The elaboration of these dyes with diverse chemical moieties is enabling the precise interrogation of biochemical and biological systems. The importance of fluorescence-based technologies in chemical biology elicits a necessity to understand the major classes of small-molecule fluorophores. Here, we examine the chemical and photophysical properties of oft-used fluorophores and highlight classic and contemporary examples in which utility has been built upon these scaffolds.

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Bright Ideas for Chemical Biology

Publisher Summary This chapter describes many of the techniques employed to study water–macromolecules interactions, their general usefulness, points out areas of mutual support and contradiction. The chapter also presents hydration of other macromolecules and the hydration of small molecules. Four broadly based approaches to operational definitions of hydration include—(1) preferential hydration, (2) hydrodynamic hydration, (3) structural hydration, and (4) low temperature hydration. The use of high resolution X-ray and neutron diffraction data on protein crystals provides an independent approach for estimation of hydration. The chapter presents several proposals for predicting protein hydration based on the amino acid composition of the protein; however, the two main questions concerned include—whether ionic groups are more hydrated than other polar groups, and whether the amide and peptide functions are hydrated or not. Low pH, and to a much smaller degree, high pH should dehydrate proteins. Intermolecular association via ionic mechanisms should be strongly dehydrating; whereas hydrophobic interactions should not have much effect on hydration levels. Although, there is a presumption that both the enthalpy and the entropy are negative for the process of adding water molecules to isolated proteins, which are maintained in their native conformations, but there is no unequivocal evidence for this.

Hydration of Proteins and Polypeptides

Unimolecular and bimolecular hybridization probes for the detection of nucleic acid target sequences comprise a target complement sequence, an affinity pair holding the probe in a closed conformation in the absence of target sequence, and either a label pair that interacts when the probe is in the closed conformation or, for certain unimolecular probes, a non-interactive label. Hybridization of the target and target complement sequences shifts the probe to an open conformation. The shift is detectable due to reduced interaction of the label pair or by detecting a signal from a non-interactive label. Certain unimolecular probes can discriminate between target and non-target sequences differing by as little as one nucleotide. Also, universal stems and kits useful for constructing said probes. Also, assays utilizing said probes and kits for performing such assays. Also, such probes capable of allelic discrimination.

Detectably labeled dual conformation oligonucleotide probes, assays and kits

Fluorescence polarization was first applied in biochemistry almost 6 decades ago, when Gregorio Weber described his studies on bovine serum albumin and ovalbumin conjugated with 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl chloride)1,2 (Figure 1). (For an overview of Gregorio Weber's wide-ranging contributions to fluorescence see3). Polarization methods became increasingly popular during the decades following Weber's work. During the past few decades, however, the increase in the number and diversity of fluorescence polarization studies has been astonishing and the method is now extremely wide-spread in the clinical and biomedical fields. The virtual explosion of polarization studies, which began during the mid-1980's, was due to several factors, including the availability of commercial instruments equipped with polarizers, the commercial availability of a great many fluorescence probes (largely due to the company Molecular Probes – now part of Invitrogen) and, in the clinical chemistry area, the introduction of the TDx instrument (and associated reagents) by Abbott Laboratories. The reasons for the popularity of fluorescence polarization in clinical and high-throughput assays are manifold. Firstly, polarization assays are homogeneous, i.e., there is no necessity for separation of free and bound ligand (these type of assays are often referred to as “mix and measure” assays). Secondly, and one of the original motivations for the development of fluorescence-based assays, there is no need for radioisotopes. Thirdly, polarization assays are reproducible and facilely automated. In this review we shall briefly trace the history of the technique and discuss in detail both theoretical and practical aspects. We shall also present copious examples from the literature, which illustrate the scope of the method in areas such as ligand binding, immunoassays, high-throughput screening, and live cell imaging and hint at future directions and applications.



Figure 1

Gregorio Weber in Hawaii – 1989. Photograph courtesy of Prof. David Jameson.




2.0 Historical Overview
As mentioned above, fluorescence polarization is widely applied in diverse fields, especially in the life sciences. A rough survey using Pubmed reveals that publications on fluorescence polarization/anisotropy numbered around a dozen in the 1950's, less than 100 in the 1960s, several hundreds in the 1970s, more than two thousand in the 1980's, more than three thousand in the1990s and close to four thousand in the 2000s to date. Given this rising trend, one may be curious how it all got started. We shall thus present a brief history of fluorescence polarization.

2.1 Discovery of Polarization
In 1808, Etienne-Louis Malus observed sunlight reflected from the windows of the Luxemburg Palace in Paris through an Iceland spar (Calcite) crystal that he rotated (Erasmus Bartholin had discovered the double refraction of light by Iceland spar in 1669). Malus discovered that the intensity of the reflected light varied as he rotated the crystal and coined the term “polarized” to describe this property of light. He published his findings in 1809 4. Malus also derived an expression for calculating the transmission of light as a function of the angle (θ) between two polarizers. This equation (Malus' Law) is now written as: I(θ) = I0 (cos2θ). Some years later, David Brewster studied the relationship between refractive index and angle of incidence on the polarization of the reflected light 5. He discovered that for normal glass and visible light, an incidence angle of ∼56 degrees resulted in total reflection of one plane of polarization – this angle is now known as Brewster's Angle. This discovery allowed Brewster to construct a polarizer composed of a “pile of plates” (interestingly, when one of the authors – DMJ – first joined Gregorio Weber's laboratory as a graduate student, Weber showed him the “pile of plates” polarizer that he had used in some of his initial studies). In 1828, William Nicol joined two crystals of Iceland spar, cut at an angle of 68°, using Canada balsam, which allowed a spatial separation between the orthogonally polarized components. Other important calcite polarizers developed around this time include: Glan-Foucault; Glan-Thompson; Glan-Taylor; Wollaston; and Rochon. Most modern spectrofluorimeters today use Glan-Taylor type calcite polarizers, which have an air-gap between the two calcite crystals allowing for transmission deep into the ultraviolet, and which makes them less susceptible to photodamage at higher irradiance levels. But the Henry Ford of polarizers was Edwin Herbert Land. In 1929 Edwin Land patented the sheet polarizer (the J-sheet), consisting of crystals of iodoquinine sulfate embedded in nitrocellulose film followed by alignment of the crystals by stretching, which led to dichroism. In 1937 he founded the Polaroid Company and in 1938 he invented the H-sheet which comprised polyvinyl alcohol sheets with embedded iodine. Land's invention made him quite rich and also allowed the use of inexpensive polarizers in diverse applications such as sunglasses and photography filters.

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Earl N. Hudson

Papers

Synthesis and characterization of two fluorescent sulfhydryl reagents

The Rotational Diffusion of Thyroglobulin A STUDY OF THYROGLOBULIN CONJUGATED WITH PYRENEBUTYRATE AND WITH DIMETHYLAMINONAPHTHALENESULFONATE