Modification of CLP with 3-OHKG Our initial studies focused on the ability of 3-OHKG to form covalent adducts with crystallins. 3-OHKG was therefore synthesized (13) and examined for its reactivity toward proteins. Following incubation with 3-OHKG, lens proteins developed coloration (365 nm) and fluorescence (Ex 380 nm/Em 490) linearly with time of incubation (Fig.1 a). Binding of the UV filter was increased approximately 10-fold if the pH was increased from 7 to 9 (Fig. 1 b), consistent with our observations on the effect of pH on GSH-3-OHKG formation (12). View larger version: Download as PowerPoint Slide Figure 1 Covalent modification of crystallins by 3-OHKG and formation of colored/fluorescent proteins that yield AHAG upon base hydrolysis. Calf lens protein was incubated with 3-OHKG for 24 days at pH 7 (a) or 8 days at pH 9. (b). UV absorbance (●) and relative fluorescence (○) are shown (a, b). Slit widths for the fluorescence measurements are Ex 10 nm/Em 5 nm (a) and Ex 5 nm/Em 5 nm (b). The fluorescence intensity on day 24, pH 7, was one-sixth that for day 8, pH 9 (Ex 380/Em 5 nm). The amount of AHAG liberated after a 48-h base hydrolysis of the samples from bis also shown in c. It is known that the amino acid side chain of kynurenine is susceptible to hydrolysis by strong base, producing an acetophenone derivative (15). In agreement with this we found that 3-OHKG, and molecules containing this moiety (i.e. GSH-3-OHKG and protein-3-OHKG adducts), are cleaved to release AHAG. This result is illustrated in Reaction FR1, where a cysteinyl adduct is given as an example and “X” and “Y” represent amino acid residues in the protein. The extent of binding of 3-OHKG to protein was therefore also assessed by base hydrolysis of the protein followed by quantification of AHAG released. Fig. 1 c shows that AHAG was released from the 3-OHKG-modified crystallins, suggesting that the covalent modification was analogous to that of the GSH-3-OHKG adduct (12). View larger version: Download as PowerPoint Slide Figure FR1

Liberation of AHAG from Human Crystallins To determine whether human crystallins are also covalently modified by 3-OHKGin vivo, lenses of various ages were examined, and the quantity of AHAG released from the proteins was plotted as a function of age (Fig. 2). AHAG has been isolated previously from cataractous lenses following treatment with base, although its origin was not investigated (16). In the present work, an age-dependent increase in AHAG was observed that was more pronounced in lenses over 40 years old, although considerable scatter was evident (Fig. 2). The inset in Fig. 2 illustrates that, in the case of GSH-3-OHKG, base hydrolysis is complete within 48 h with quantitative recovery. More than half (85%) of the AHAG was released from the modified crystallins within 48 h (Fig. 2,inset). Duplicate samples of 22 of the lenses over 40 years old were also incubated for 144 h. This action resulted in a 15% increase in AHAG detected, on average, but did not reduce the variability (scatter) of the data depicted in Fig. 2. Significant variation in the extent of UV filter-mediated modification of crystallins do, therefore, exist in our study population. We estimate, based on the molar absorptivity of GSH-3-OHKG (12) compared with the known increase in lenticular absorption of light at 360 nm (1), that at least 50% of the increase in age-related lenticular color in humans may be attributed to binding of 3-OHKG. View larger version: Download as PowerPoint Slide Figure 2 AHAG liberated from human lens proteins as a function of age. Fifty-five lenses were extracted with 80% (v/v) ethanol/water four times to remove unbound UV filters. The lyophilized protein was incubated for 48 h in 5% (w/v) potassium hydroxide in 80% (v/v) ethanol to liberate AHAG, which was quantified by HPLC. Theinset shows time course experiments for the base hydrolysis of the GSH-3-OHKG adduct (▪) and lens proteins (○). In the latter case, the data are means with the error bars showing S.E., where n = 2, 3, 22, 3, and 22 samples for the times 0.5, 1, 2, 4, and 6 days, respectively. The percentage recovery of AHAG from lens proteins was shown to increase by approximately 15% when the incubation time was increased from 2 to 6 days. For reasons of practicality, the 48-h incubation was adopted as the routine hydrolysis procedure. Confirmation of the structure of liberated AHAG was obtained via microbore HPLC with LC-ESIMS and by comparison with an authentic standard synthesized (14) in our laboratory. Fig.3 shows that the AHAG released from the isolated human lens proteins displayed a positive ion atm/z 314, with a fragment ion atm/z 152, consistent with the molecular mass of AHAG (313 Da) and its aglucone (151 Da). This mass spectrum was identical to the synthetic AHAG (Fig. 3). The lens-derived AHAG and the synthetic standard also co-eluted when they were mixed together and analyzed by LC-ESIMS. These data provide the first chemical evidence that 3-OHKG forms adducts with human lens proteins according to the mechanism we have proposed, which involves addition at the βC of the side chain (see Reaction FR1). View larger version: Download as PowerPoint Slide Figure 3 Mass spectra of synthetic and lens protein-derived AHAG . The AHAG was either derived from base hydrolysis of human lens crystallins (a) or synthesized (b) as described under “Experimental Procedures.” Samples were analyzed using microbore HPLC with in-line electrospray ionization mass spectrometry. The positive ion mass spectra shown are for the single 360 nm-absorbing compounds present and were determined directly after elution from the HPLC column. The y ordinates show relative signal intensity.