Separation of multiple yellow fluorescent lipofuscin components in rat kidney and their characterization

Yellow fluorescent lipofuscin deposited in rat kidney was extracted in an aqueous solution and characterized after separation. Centrifugal fractionation of the extract revealed that most of the yellow fluorescence was detected in the 105,000 x g-supernatant, and little in nuclei, cell debris, mitochondria, lysosomes, microsomes and plasma membrane. The yellow fluorescence in the supernatant was fractionated by gel filtration through Sephadex columns into 5 yellow fluorescent fractions A, B (B1, B2 and B3) and C showing the same fluorescence spectra with excitation maximum/emission maximum at 400/620 nm. The components in fraction A were converted into the smaller molecular-weight components in fraction B on treatment with 4 M urea or protease, suggesting that they were proteinaceous. The smallest molecular-weight fluorescent components in fraction C were adherent to solid cellulose materials. The fluorescent components in all the fractions were soluble in water and insoluble in chloroform-methanol, indicating that they were not lipidic materials. The fluorophores in these fractions were kept stable on borohydride treatment, but readily converted into non-fluorescent components on heavy-metal ion treatment. The characteristics of the yellow fluorescence in these fractions were quite different from those of bluish lipofuscin-like fluorophores that may be generated in tissues during lipid peroxidation.