Frequency domain fluorometry: theory and application.

Frequency domain fluorometry is a widely utilized tool in the physical, chemical, and biological sciences. This chapter focuses on the theory of the method and the practical aspects required to carry out intensity decay, i.e., lifetime measurements on a modern frequency domain fluorometer. Several chemical/biological systems are utilized to illustrate data acquisition protocols. Data analysis procedures and methodologies are also discussed.

Spectroscopic investigation of the noncovalent association of the nerve agent simulant diisopropyl methylphosphonate (DIMP) with zinc(II) porphyrins.

Organophosphonates pose a significant threat as chemical warfare agents, as well as environmental toxins in the form of pesticides. Thus, methodologies to sense and decontaminate these agents are of significant interest. Porphyrins and metalloporphyrins offer an excellent platform to develop chemical threat sensors and photochemical degradation systems. These highly conjugated planar molecules exhibit relatively long-lived singlet and triplet states with high quantum yields and also form self-associated complexes with a wide variety of molecules. A significant aspect of porphyrins is the ability to functionalize the peripheral ring system either directly to the pyrrole rings or to the bridging methine carbons. In this report, steady-state absorption and fluorescence are utilized to probe binding affinities of a series of symmetric and asymmetric zinc(II) metalloporphyrins for the nerve agent simulant diisopropyl methylphosphonate (DIMP) in hexane. The red shifts in the absorption and emission spectra observed for all of the metalloporphyrins probed are discussed in the frame of Gouterman's four orbital model and a common binding motif involving coordination between the metalloporphyrin and DIMP via interaction between the zinc metal center of the porphyrin and phosphoryl oxygen of DIMP (Zn-O═P) is proposed.

Investigations of protein-protein interactions using time-resolved fluorescence and phasors.

Protein interactions are critical for biological specificity and techniques able to characterize these interactions are of fundamental importance in biochemistry and cell biology. Fluorescence methodologies have been extremely useful for studying many biological systems including protein-ligand and protein-protein interactions. In this review we focus on the application of time-resolved fluorescence approaches to macromolecular systems. We also include a detailed discussion of a relatively new time-resolved technique, the phasor method, for studying protein interactions both in vitro and in live cells.

Mimicking heme enzymes in the solid state: metal-organic materials with selectively encapsulated heme.

To carry out essential life processes, nature has had to evolve heme enzymes capable of synthesizing and manipulating complex molecules. These proteins perform a plethora of chemical reactions utilizing a single iron porphyrin active site embedded within an evolutionarily designed protein pocket. We herein report the first class of metal-organic materials (MOMs) that mimic heme enzymes in terms of both structure and reactivity. The MOMzyme-1 class is based upon a prototypal MOM, HKUST-1, into which catalytically active metalloporphyrins are selectively encapsulated in a "ship-in-a-bottle" fashion within one of the three nanoscale cages that exist in HKUST-1. MOMs offer unparalleled levels of permanent porosity and their modular nature affords enormous diversity of structures and properties. The MOMzyme-1 class could therefore represent a new paradigm for heme biomimetic catalysis since it combines the activity of a homogeneous catalyst with the stability and recyclability of heterogeneous catalytic systems within a single material.

Time resolved thermodynamics associated with ligand photorelease in heme peroxidases and globins: Open access channels versus gated ligand release.

Heme proteins represent a diverse class of biomolecules responsible for an extremely diverse array of physiological functions including electron transport, monooxygenation, ligand transport and storage, cellular signaling, respiration, etc. An intriguing aspect of these proteins is that such functional diversity is accomplished using a single type of heme macrocycle based upon iron protoporphyrin IX. The functional diversity originates from a delicate balance of inter-molecular interactions within the protein matrix together with well choreographed dynamics that modulate the heme electronic structure as well as ligand entry/exit pathways from the bulk solvent to the active site. Of particular interest are the dynamics and energetics associated with the entry/exit of ligands as this process plays a significant role in regulating the rates of heme protein activity. Time-resolved photoacoustic calorimetry (PAC) has emerged as a powerful tool through which to probe the underlying energetics associated with small molecule dissociation and release to the bulk solvent in heme proteins on time scales from tens of nanoseconds to several microseconds. In this review, the results of PAC studies on various classes of heme proteins are summarized highlighting how different protein structures affect the thermodynamics of ligand migration. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.

Excited state properties of 9-amino acridine surface adsorbed onto αZr-phosphate particles.

In this report the photo-physical properties of 9-amino acridine (9AA) associated with αZr-phosphate particles (αZrP) is examined. In ethanol solution 9AA exhibits absorption maxima at 425 nm, 402 nm and 383 nm as well as emission bands centered at 455 nm and 483 nm (using 423 nm excitation). The corresponding emission decay is monophasic with a lifetime of 16.5 ns. When αZrP is sonicated in the presence of an ethanol solution of 1 mM 9AA the resulting material exhibits a broad absorption band centered at ∼400 nm with shoulders at ∼425 nm and ∼385 nm and emission bands at 462 nm and 485nm (using 423 nm excitation). Interestingly, the emission decay is biphasic with lifetimes of 1.6 ns and 9.8 ns constituting 57% and 43% of the total emission intensity, respectively. The absence of any shifts in the low angle XRD data suggests that 9AA associates via adsorption onto the exterior surfaces of the αZrP particles. Overall, these results are consistent with different modes of 9AA association to αZrP reflecting different degrees of H-bonding which significantly influences the extent of non-radiative decay from the lowest excited singlet state of 9AA.