PHOTOCHEMISTRY OF TYPE I ACID-SOLUBLE CALF SKIN COLLAGEN: DEPENDENCE ON EXCITATION WAVELENGTH

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 (335–400nm) and broad-band solar-simulating radiation (SSR; 290^1–00nm). 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 at430–435 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 at400–420 nm, attributable to dityrosine, forms under UVC, but not under solar radiation. This band is photolabile to UVA and UVB wavelengths. Amino acid analysis indicates significant destruction of aromatic amino acids under UVC, but not under UVA or SSR. When properly understood, collagen fluorescence fading phenomena may act as a sensitive molecular probe of structure, conformation and reactivity.