Lysozyme Photochemistry as a Function of Temperature. The Protective Effect of Nanoparticles on Lysozyme Photostability

Abstract

The presence of aromatic residues and their close spatial proximity to disulphide bridges makes hen egg white lysozyme labile to UV excitation. UVB induced photo-oxidation of tryptophan and tyrosine residues leads to photochemical products, such as, kynurenine, N-formylkynurenine and dityrosine and to the disruption of disulphide bridges in proteins. We here report that lysozyme UV induced photochemistry is modulated by temperature, excitation power, illumination time, excitation wavelength and by the presence of plasmonic quencher surfaces, such as gold, and by the presence of natural fluorescence quenchers, such as hyaluronic acid and oleic acid. We show evidence that the photo-oxidation effects triggered by 295 nm at 20°C are reversible and non-reversible at 10°C, 25°C and 30°C. This paper provides evidence that the 295 nm damage threshold of lysozyme lies between 0.1 μW and 0.3 μW. Protein conformational changes induced by temperature and UV light have been detected upon monitoring changes in the fluorescence emission spectra of lysozyme tryptophan residues and SYPRO® Orange. Lysozyme has been conjugated onto gold nanoparticles, coated with hyaluronic acid and oleic acid (HAOA). Steady state and time resolved fluorescence studies of free and conjugated lysozyme onto HAOA gold nanoparticles reveals that the presence of the polymer decreased the rate of the observed photochemical reactions and induced a preference for short fluorescence decay lifetimes. Size and surface charge of the HAOA gold nanoparticles have been determined by dynamic light scattering and zeta potential measurements. TEM analysis of the particles confirms the presence of a gold core surrounded by a HAOA matrix. We conclude that HAOA gold nanoparticles may efficiently protect lysozyme from the photochemical effects of UVB light and this nanocarrier could be potentially applied to other proteins with clinical relevance. In addition, this study confirms that the temperature plays a critical role in the photochemical pathways a protein enters upon UV excitation.

Fig 1. LYZ molecular structure, according to (2LYZ.pdb).

Aromatic residues are represented by different colors: Trp (red), Tyr (blue), Cys (yellow).

Fig 2. LYZ thermal ramp and data first derivative from 45–90°C (T_m = 74°C), with heating rate fixed at 1°C/min.

Fluorescence excitation was fixed at 295 nm and fluorescence emission at 350 nm and excitation slit was set at 0.1 mm (0.1 μW).

Fig 3. LYZ fluorescence excitation and emission spectra, before and after the thermal ramp at 45–90°C.

Fluorescence excitation was fixed at 295 nm and fluorescence emission at 350 nm and excitation slit was set at 0.1 mm (0.1 μW).

Fig 4. Continuous 295 nm excitation of LYZ at 20°C, for 2 hours.

Fluorescence emission at 330 nm and excitation slit was set at 0.1 mm (0.1 μW).

Fig 5. LYZ fluorescence excitation and emission spectra, before and after 295 nm continuous excitation for 2 hours.

Fluorescence excitation was fixed at 295 nm and fluorescence emission at 330 nm and excitation slit was set at 0.1 mm (0.1 μW).

Fig 6

A) LYZ 2 hours excitation from 250 nm until 310 nm, at 20°C. Fluorescence emission wavelength was fixed at 330 nm, while excitation wavelengths were selected as following: at 250 nm (light blue), at 265 nm (dark blue), at 285 nm (light green), at 295 nm (dark green), at 305 nm (dark grey) and at 310 nm (light grey). B) Closer look at LYZ 2hours excitation at 250 nm, 305 nm and 310 nm, at 20°C. For all excitation wavelengths, fluorescence emission wavelength was fixed at 330 nm and slit fixed at 0.5 mm (1.0 μW).

Fig 7

A) Monitoring LYZ with SYPRO^® Orange spectra before and after 70 min 295 nm excitation of LYZ at 20°C, with excitation slit fixed at 0.5 mm (1.0 μW). Fluorescence intensity of SYPRO^® Orange was fixed at 580 nm and excitation at 470 nm. B) SYPRO^® Orange emission intensity (excitation wavelength fixed at 470 nm) for every 10 min of LYZ 295 nm excitation for 70 min.

Fig 8. LYZ fluorescence 295 nm 2hours excitation at 20°C, using different excitation slit openings: 0.1 mm (0.1 μW), 0.3 mm (0.5 μW), 0.4 mm (0.7 μW) and 0.5 mm (1.0 μW).

Fluorescence emission wavelength was fixed at 330 nm. At the upper corner is displayed the equation of the dependence of the excitation slit size (x) versus excitation power (y).

Fig 9

A) LYZ fluorescence excitation and emission spectra before and after LYZ 295 nm 2hours excitation, with slit opening fixed at 0.1 mm (0.1 μW). B) LYZ fluorescence excitation and emission spectra before and after LYZ 295 nm 2hours excitation, with slit opening fixed at 0.5 mm (power: 1.0 μW). Fluorescence excitation was fixed at 295 nm and fluorescence emission at 330 nm.

Fig 10. Temperature effect on LYZ photochemistry: at 10°C (light blue), 20°C (dark blue), 25°C (light grey) and 30°C (dark grey), for four open/close cycles of periods of 10 min of excitation followed by 10 min in the dark.

Fluorescence excitation and emission wavelengths were fixed at 295 nm and 330 nm, respectively. Excitation slit size was set at 0.5 mm (1.0 μW) for all experiments.

Fig 11. SYPRO^® Orange emission intensity spectra before and after LYZ 295 nm excitation at different temperatures (10°C, 20°C, 25°C and 30°C).

SYPRO^® fluorescence excitation wavelength was fixed at 470 nm and acquired before and after the four open/close cycles. Excitation slit size was set at 0.5 mm (1.0 μW).

Fig 12

A) LYZ fluorescence emission intensity at 330nm monitored at 20°C; and B) at 10°C. Samples are described as: a) fresh LYZ never previously illuminated, b) LYZ after 30 min of continuous 295 nm illumination, c) LYZ after 30 min of continuous 295 nm illumination followed by 48 hours in the dark, d) LYZ after 30 min of continuous 295 nm illumination followed by 48 hours in the dark and subsequent further 30 min of continuous 295 nm. LYZ excitation was fixed at 295 nm with a slit size was set at 0.5 mm (1.0 μW).

Fig 13. LYZ fluorescence emission intensity at 330 nm for free LYZ (2 h 295 nm excitation), LYZ-conjugated HAOA GNPs (2 h 295 nm excitation), and empty HAOA gold nanoparticles (GNPs) and non-coated plain GNPs (1h 295 nm excitation).

All samples were analyzed at 20°C and excitation slit size fixed at 2.0 mm (4.4 μW). At the upper corner, LYZ-conjugated HAOA GNPs excited for 2 hours is compared to HAOA GNPs and plain GNPs.

Fig 14

A) Representative illustration of LYZ-conjugated HAOA coated gold nanoparticles; B) TEM image of HAOA coated gold nanoparticles (non-conjugated) at scale bar: 100 nm; and C) Intensity analysis of the HAOA gold nanoparticles TEM image.

Fig 15. Conjugation effect: LYZ in supernatant (after conjugation) compared with free LYZ and LYZ-conjugated HAOA gold nanoparticles (GNPs).

Fluorescence excitation spectra was fixed at 330 nm and fluorescence emission spectra was fixed at 295 nm. Experiments were conducted at 20°C and excitation slit size fixed at 2.0 mm (4.4 μW). LYZ was not continuously excited and only the necessary UV light was used for obtaining the displayed spectra.

Fig 16

A) Fluorescence emission spectra for NFK + Kyn, before and after excitation of free LYZ and LYZ-conjugated HAOA gold nanoparticles (GNPs), at a fixed wavelength of 320 nm. Experiments were conducted at 20°C and excitation slit size fixed at 2.0 mm (4.4 μW); B) Fluorescence emission spectra for Kyn + NFK, before and after excitation of free LYZ and LYZ-conjugated HAOA gold nanoparticles (GNPs), at a fixed wavelength of 360 nm. Experiments were conducted at 20°C and excitation slit size fixed at 2.0 mm (4.4 μW).