Abstract
Recently, the photoexcited triplet state of porphyrin was proposed as a promising spin-label for pulsed dipolar electron paramagnetic resonance (EPR). Herein, we report the factors that determine the electron spin echo dephasing of the photoexcited porphyrin in a water–glycerol matrix. The electron spin relaxation of a water-soluble porphyrin was measured by Q-band EPR, and the temperature dependence and the effect of solvent deuteration on the relaxation times were studied. The phase memory relaxation rate (1/Tm) is noticeably affected by solvent nuclei and is substantially faster in protonated solvents than in deuterated solvents. The Tm is as large as 13–17 μs in deuterated solvent, potentially expanding the range of distances available for measurement by dipole spectroscopy with photoexcited porphyrin. The 1/Tm depends linearly on the degree of solvent deuteration and can be used to probe the environment of a porphyrin in or near a biopolymer, including the solvent accessibility of porphyrins used in photodynamic therapy. We characterized the noncovalent binding of porphyrin to human serum albumin (HSA) from 1/Tm and electron spin echo envelope modulation (ESEEM) and found that porphyrin is quite exposed to solvent on the surface of HSA. The 1/Tm and ESEEM are equally effective and provide complementary methods to determine the solvent accessibility of a porphyrin bound to protein or to determine the location of the porphyrin.
Highlights
Pulsed dipolar electron paramagnetic resonance (PD EPR) is a powerful technique for nanoscale distance measurements in biomolecules, including large, disordered systems [1,2,3,4,5,6]
The common spin labels used in PD EPR
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Summary
Pulsed dipolar electron paramagnetic resonance (PD EPR) is a powerful technique for nanoscale distance measurements in biomolecules, including large, disordered systems [1,2,3,4,5,6]. The dipole–dipole interaction between two spin labels attached to the biomolecule is measured as a periodic modulation of an EPR signal. This interaction scales with the distance, r, between the labels as 1/r3 , providing accurate values for the average distance between the labels and even the distribution of distances from the different structural conformations present in the sample. The photoexcited triplet states of porphyrin [12,13,14,15] and fullerene [16]
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