Abstract
Abstract: :
Purpose: Isolation and identification of chromophores and fluorophores from enzymatic digests of normal human lens protein, and characterization of their age-related variation and possible pathways for their formation. Method: Lens water-soluble and insoluble protein fractions were isolated from normal human lenses of different age. Each protein fraction was digested with a battery of proteolytic enzymes under argon and then subjected to Bio-Gel P-2 (1 x 25 cm) size-exclusion chromatography. A late eluting hydrophobic peak (P 5) was collected and then fractionated by semi-preparative prodigy C-18 RP-HPLC. TLC, spectral analysis and LC-MS analysis further characterized the peaks obtained from P 5 by HPLC. Furthermore, sodium borohydride and DTPA treatments were used to clarify the possible origin of kynurenine and anthranilic acid in the lens protein digests. Results: A higher level of peak 5 (P 5) was observed in the water-soluble fraction compared to the water-insoluble fraction by gel filtration chromatography. By RP-HPLC peak 5 from the water-soluble fraction was resolved into two peaks (P5-1 and 2), while three peaks were seen in the peak 5 from the water-insoluble fraction (P 5-1, 2, 3). TLC, HPLC and LC-MS confirmed that peaks 5-1 and 2 from both sources were kynurenine and anthranilic acid respectively. Peak 5-3 exhibited an absorbance maximum at 314 nm and a molecular weight of 407, however, its identity remains unknown. Both kynurenine and anthranilic acid were markedly increased in digests from the water-insoluble fraction from aged human lens, but neither were present in a digest of calf lens proteins. Treatment of the human lens proteins with NaBH4 reduced the kynurenine release. The water-insoluble digest from aged human lens converted added kynurenine to anthranilic acid, but this oxidation was prevented by the addition of the chelating agent, DTPA. Conclusion: The presence of kynurenine and anthranilic acid were confirmed by HPLC, TLC and LC-MS analyses in enzymatic digests of human lens protein and their age-dependent variation was characterized. Preliminary data suggest that kynurenine is bound to proteins in part by a Schiff base linkage. Anthranilic acid appears to result from a transition metal-catalyzed oxidation of kynurenine released during the digestion.
Keywords: 309 aging • 504 oxidation/oxidative or free radical damage • 525 protein modifications-post translational