Quantitative Determination of Singlet Oxygen Generated by Excited State Aromatic Amino Acids, Proteins, and Immunoglobulins

Abstract

Singlet oxygen, 1 O 2 , is a highly reactive electronically excited state of oxygen invoked in many physiological and pathological processes. 1 While its generation in biological systems is mainly traced back to photosensitization by sunlight-absorbing cofactors and dark enzymatic pathways, 1 O 2 can also form by photosensitization with aromatic amino acids such as tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe), which are abundant light absorbers in the UV-B range (290-320 nm). 2 Notably, though the role of 1 O 2 in the formation of H 2 O 2 by in vitro UV irradiation of aromatic amino acids in immunoglobulins was reported by Wentworth et al., 3 the generation of 1 O 2 by aromatic amino acids and biological macromolecules has not been analyzed quantitatively. 4 In this manuscript we report the quantum yields of 1 O 2 generation upon excitation of Trp, Tyr, and Phe in their zwitterionic forms, as methyl esters, and within a few test proteins and immunoglobulins.The sensitized generation of 1 O 2 starts by excitation of the amino acid by absorption of UV light, followed by intersystem crossing to the triplet state (eqs 1 and 2), and is completed by energy transfer to oxygen in its triplet ground state (eq 3). The quantum yields of 1 O 2 can be obtained in air-saturated solutions by measuring the quantum yield of its near IR emission (eq 4 and Figure 1).The amino acids Phe, Trp, and Tyr, the N-acetylated amino acids NAc-Phe, NAc-Trp, and NAc-Tyr, the proteins bovine serum albumin (BSA) and ovoalbumin (OVA), and the immunoglobulins bovine-IgG, human-IgG, and sheep-IgG were dissolved in either D 2 O or MeCN. Samples were irradiated with a 266 nm pulse from a frequency quadrupled continuum Nd:YAG laser (8 ns, 3 mJ).The near-IR luminescence from O 2 ( 1 ∆ g ) was collected at right angles to excitation after focusing onto the variable slit of a SPEX 1681 monochromator and detected using a Hamamatsu PMT sensitive in the near-IR region. The near-IR luminescence spectrum of singlet oxygen photosensitized by phenylalanine in an air-saturated D 2 O solution is shown in Figure 1. There is a strong peak at about 1270 nm, characteristic of singlet oxygen phosphorescence. A time-resolved decay trace of the observed phosphorescence has a lifetime (τ ∆ ) of about 48 µs, consistent with reported values of 1 O 2 in D 2 O. Solutions of tryptophan, tyrosine, bovine serum albumin (BSA), ovalbumin (OVA), the immunoglobulins bovine IgG, human IgG, and sheep IgG in D 2 O, and the N-acetyl amino acids in MeCN were irradiated under similar conditions to emit spectra consistent with singlet oxygen luminescence. Argon-purged solutions of each compound showed no near-IR luminescence.The quantum yields of oxygen (Φ ∆ ) were determined using methylene blue in D 2 O (Φ ∆ ) 0.52) or MeCN (Φ ∆ ) 0.52) as a reference. 5 Triplicate kinetic analysis of decay traces for Tyr and Trp gave lifetimes for singlet oxygen of 35-40 µs in D 2 O, which are about 20% shorter than those observed with Phe. This may be attributed to quenching, in agreeme...

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