Photo-isomerization of the isolated photoactive yellow protein chromophore: what comes before the primary step?

Photoactive proteins typically rely on structural changes in a small chromophore to initiate a biological response. While these changes often involve isomerization as the "primary step", preceding this is an ultrafast relaxation of the molecular framework caused by the sudden change in electronic structure upon photoexcitation. Here, we capture this motion for an isolated model chromophore of the photoactive yellow protein using time-resolved photoelectron imaging. It occurs in <150 fs and is apparent from a spectral shift of ∼70 meV and a change in photoelectron anisotropy. Electronic structure calculations enable the quantitative assignment of the geometric and electronic structure changes to a planar intermediate from which the primary step can then proceed.

A study of hydrogen peroxide chemistry and photochemistry in tea stain solution with relevance to clinical tooth whitening.

OBJECTIVE: Tooth whitening using hydrogen peroxide is a complex process, and there is still some controversy about the roles of pH, temperature, chemical activators, and the use of light irradiation. In this work the basic interactions between whitening agents and stain molecules are studied in simple solutions, thus avoiding the physics of diffusion and light penetration in the tooth to give clarity on the basic chemistry which is occurring. METHOD: The absorbance of tea stain solution at 450 nm was measured over a period of 40 min, with various compositions of whitening agent added (including hydrogen peroxide, ferrous gluconate and potassium hydroxide) and at the same time the samples were subjected to blue light (465 nm) or infra-red light (850 nm) irradiation, or alternatively they were heated to 37°C. RESULTS: It is shown that the reaction rates between chromogens in the tea solution and hydrogen peroxide can be accelerated significantly using ferrous gluconate activator and blue light irradiation. Infra red irradiation does not increase the reaction rate through photochemistry, it serves only to increase the temperature. Raising the temperature leads to inefficiency through the acceleration of exothermic decomposition reactions which produce only water and oxygen. CONCLUSION: By carrying out work in simple solution it was possible to show that ferrous activators and blue light irradiation significantly enhance the whitening process, whereas infra red irradiation has no significant effect over heating. The importance of controlling the pH within the tooth structure during whitening is also demonstrated.

Electron photodetachment dissociation of DNA anions with covalently or noncovalently bound chromophores.

Double stranded DNA multiply charged anions coupled to chromophores were subjected to UV-Vis photoactivation in a quadrupole ion trap mass spectrometer. The chromophores included noncovalently bound minor groove binders (activated in the near UV), noncovalently bound intercalators (activated with visible light), and covalently linked fluorophores and quenchers (activated at their maximum absorption wavelength). We found that the activation of only chromophores having long fluorescence lifetimes did result in efficient electron photodetachment from the DNA complexes. In the case of ethidium-dsDNA complex excited at 500 nm, photodetachment is a multiphoton process. The MS(3) fragmentation of radicals produced by photodetachment at lambda = 260 nm (DNA excitation) and by photodetachment at lambda > 300 nm (chromophore excitation) were compared. The radicals keep no memory of the way they were produced. A weakly bound noncovalent ligand (m-amsacrine) allowed probing experimentally that a fraction of the electronic internal energy was converted into vibrational internal energy. This fragmentation channel was used to demonstrate that excitation of the quencher DABSYL resulted in internal conversion, unlike the fluorophore 6-FAM. Altogether, photodetachment of the DNA complexes upon chromophore excitation can be interpreted by the following mechanism: (1) ligands with sufficiently long excited-state lifetime undergo resonant two-photon excitation to reach the level of the DNA excited states, then (2) the excited-state must be coupled to the DNA excited states for photodetachment to occur. Our experiments also pave the way towards photodissociation probes of biomolecule conformation in the gas-phase by Förster resonance energy transfer (FRET).

Modest in vivo exposure to solar wavelengths induces a visible absorbing chromophore in Skh-1 mouse epidermis.

BACKGROUND/PURPOSE: In the previous work, we correlated epidermal hyperplasia with increased epidermal absorption in the 250-400 nm region. During a recent review of that work, the apparent formation of a chromophore, with absorption slightly longer than 400 nm, in the epidermis of irradiated animals was noted. In this study, we have extended the transmission measurement to include the 250-800 nm region. METHODS: Age-matched Skh-1 hairless mice were separated into three groups. One group was irradiated with 6.3 J/cm(2) (0.9 minimal erythemal dose; MED) of solar simulating ultraviolet radiation (SSUVR) five times/week for 2 weeks, then increased to 1.1 MED (7.1 J/cm(2)) for two additional weeks (20-day group). A second 10-day group, added halfway through the protocol, was irradiated with 0.9 MED five times/week for 2 weeks. The control group received no UV irradiation. Routine H&E staining and epidermal absorption spectral analysis were carried out on biopsy specimens from each animal. RESULTS: This work confirms the development or enhancement of a visible chromophore with a maximum absorption at ca 412 nm. This peak appears to be radiation dose dependent. It can be discerned in both the groups, albeit more prominently in the 20-day animals. The absorption is sufficiently strong to impart a yellow to reddish appearance to skin viewed in full spectrum visible light. CONCLUSIONS: Accumulation of such a chromophore in humans may contribute to the coloration of chronically exposed skin. The absorption strength and wavelength location of the peak is strongly suggestive of a heme-like compound. We are currently conducting experiments to further characterize this chromophore.

Lowry method of protein quantification: evidence for photosensitivity.

Despite reports of its susceptibility to various interfering factors, the Folin Phenol protein quantification method of O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall (1951, J. Biol. Chem. 193, 265-275) remains the most convenient and accurate method for routine protein determinations. Our findings indicate that the Lowry assay is also photosensitive which can result in a discrepancy of up to 10% in estimated protein concentrations, unless appropriate precautions are taken.