Temperature dependence of photochemical fluorescence fading in Skh‐1 hairless mouse collagen

Abstract

Summary Background Type I mammalian collagens have several photolabile fluorescent moieties that absorb UV rays capable of reaching the dermis. We studied the temperature dependence of fluorescence fading as a marker of photochemical damage. Methods Collagen solutions were exposed to radiation from 0 to 240 min from either a UVG-11 hand lamp, total dose = 1.173 × 103 J/m2; a UVL-21 hand lamp total dose = 2.030 × 103 J/m2; or the fluorometer, at 325 ± 5 nm, total dose = 0.156 × 103 J/m2. We recorded intensities at excitation/emission wavelengths 270/300, 270/330, 270/360, 270/400, 325/400, and 370/450 nm at T = 9.0–59.3 1°C. Results Results indicated simultaneous forward and reverse reactions. However, the 270/360 nm fluorophore could be analyzed as a second-order reaction. The Arrhenius curve showed two straight lines intersecting near the denaturation temperature, with helix activation energy Ea~0 and coil Ea = 7.6 ± 0.6 kcal/mol (31.7 ± 2.5 kJ/mol). Discussion Collagen-bound fluorophores are not just passive markers of oxidative stress and age-related damage. Their photolability to wavelengths reaching the dermis may result in pathological conditions, particularly at elevated body temperatures.

Thermal and Photochemical Effects on the Fluorescence Properties of Type I Calf Skin Collagen Solutions at Physiological pH

“…The second order fading of the (270)360 nm species suggests a "double molecule", which we have previously attributed to an "excimer-like" interaction between two tyrosine molecules in close proximity [12]. Alternatively, it may be a stable, as yet uncharacterized, DOPA oxidation product.…”

“…Changes in the other fluorescence pairs were kinetically complicated alterations and were not amenable to simple analysis (see [12] for results with Skh-1 hairless mouse collagen).…”

“…Readings were taken at excitation/emission wavelengths of 270/300 (tyrosine), 270/330 (DOPA), 270/360 ("interacting" tyrosine or DOPA oxidation product(s)), 270/400 (link between excitation at 270 nm and 325 nm), 325/400 (dityrosine), and 370/450 nm (AGE's) (not relevant here); DOPA oxidation product(s) [12] [13]. The choice of excitation/emission wavelength pairs was chosen to limit interference from overlapping bands.…”

“…Photo-induced dityrosine formation and fluorescence disappearance ("fading") of DOPA oxidation products ("oxidation products") attending exposure to UV radiation have been used as convenient markers of the extent of photolysis [11] [12]. This photolability suggests the possibility of collagen or its oxidation products acting as in situ photosensitizers and/or phototoxic agent.…”

Fluorescence Lifetime Measurements and Biological Imaging

“…Both collagen and elastin fluoresce under UV and blue excitation; collagen shows a broad emission up to 500 nm, depending on the excitation wavelength (270-370 nm). 291 Blue emission of collagen was attributed to tyrosine and dityrosine, 292,293 while the yellow-green fluorescence was assigned to pentosidine (Figure 28), formed in vivo via a Maillard reaction involving pentose lysine and arginine. 294 (The same Maillard reaction is actually responsible for bread darkening in toasters and making beer golden brown).…”