Photochemistry of Type I Acid‐soluble Calf Skin Collagen: Dependence on Excitation Wavelength

Abstract

Although previous studies have demonstrated that the predominant photochemistry of type I collagen under 254 nm irradiation may be attributed either to direct absorption by tyrosine/phenylalanine or to peptide bonds, direct collagen photochemistry via solar UV wavelengths is much more likely to involve several age- and tissue-related photolabile collagen fluorophores that absorb in the latter region. In this study, we compare and contrast results obtained from irradiation of a commercial preparation of acid-soluble calf skin type I collagen in solution with UVC (primarily 254 nm), UVA (335-400 nm) and broad-band solar-simulating radiation (SSR; 290-400 nm). Excitation spectroscopy and analysis of photochemically induced disappearance of fluorescence (fluorescence fading) indicates that this preparation has at least four photolabile fluorescent chromophores. In addition to tyrosine and L-3,4-dihydroxyphenylalanine, our sample contains two other fluorophores. Chromophore I, with emission maximum at 360 nm, appears to be derived from interacting aromatic moieties in close mutual proximity. Chromophore II, with broad emission at 430-435 nm, may be composed of one or more age-related molecules. Collagen fluorescence fading kinetics are sensitive to excitation wavelength and to conformation. Under UVC, chromophore I fluorescence disappears with second-order kinetics, indicating a reaction between two proximal like molecules. Adherence to second-order kinetics is abrogated by prior denaturation of the collagen sample. A new broad, weak fluorescence band at 400-420 nm, attributable to dityrosine, forms under UVC, but not under solar radiation. This band is photolabile to UVA and UVB wavelengths.(ABSTRACT TRUNCATED AT 250 WORDS)

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

“…However comparison of these spectra with that of pure tyrosine [14] shows a slight broadening of the overall fluorescence envelope, due to small amounts of DOPA and its oxidation products (Figure 2). The older sample, #072005, shows significant diminution of the tyrosine peak at 305 nm (I (072005) /I (072012) = 0.57) as well as the more obvious appearance of additional longer wavelength bands that can be ascribed to a mixture of DOPA, DOPA oxidation products and dityrosine [11]- [13].…”

“…Both r 1 and r 2 are proportional to fluorophore concentration [9] [11]. 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).…”

“…The resulting fluorescence intensity, I f (t) vs. irradiation time t plots were analyzed kinetically. The 270/360 nm fluorescence fading was analyzed as a second-order plot of 1/I(t) vs. t to afford a second order rate parameter [11] r 2 , whose slope is proportional to a molecular rate constant for disappearance of the (unknown) fluorophore at 270/360 nm, is:…”

“…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.…”

The thermal behavior of collagen in solution: Effect of glycerol and 2-propanol

“…The shoulder peak at 280 nm might derive from phenylalanine [32]. The peak at ∼360 nm was attributed to an excimer-like species, occurring as a result of interaction between two tyrosine residues in close mutual proximity [33] and the peak at ∼410 nm was corresponded to bityrosine, which could be formed photochemically from neighboring tyrosine residues [34]. Fig.…”