P.R. Olesen

Ascorbic acid-induced crosslinking of lens proteins: evidence supporting a Maillard reaction

The incubation of calf lens extracts with 20 mM ascorbic acid under sterile conditions for 8 weeks caused extensive protein crosslinking, which was not observed with either 20 mM sorbitol or 20 mM glucose. While no precipitation was observed, ascorbic acid did induce the formation of high-molecular-weight protein aggregates as determined by Agarose A-5m chromatography. Proteins modified by ascorbic acid bound strongly to a boronate affinity column, however, crosslinked proteins were present mainly in the unbound fraction. These observations suggest that the cis-diol groups of ascorbic acid were present in the primary adduct, but were either lost during the crosslinking reaction or sterically hindered from binding to the column matrix. The amino acid composition of the ascorbic acid-modified proteins was identical to controls except for a 15% decrease in lysine. Amino acid analysis after borohydride reduction, however, showed a 25% decrease in lysine, a 7% decrease in arginine and an additional peak which eluted between phenylalanine and histidine. Extensive browning occurred during the ascorbic acid-modification reaction. This resulted in protein-bound chromophores with a broad absorption spectrum from 300 to 400 nm, and protein-bound fluorophores with excitation/emission maxima of 350/450 nm. A 4 week incubation of dialyzed crude lens extract with [1-14C]ascorbic acid showed increased incorporation for 2 weeks, followed by a decrease over the next 2 weeks as crosslinking was initiated. The addition of cyanoborohydride to the reaction mixture completely inhibited crosslinking and increased [1-14C]ascorbic acid incorporation to a plateau value of 180 nmol per mg protein. Amino acid analysis showed a 50% loss of lysine, and 8% decrease in arginine and the presence of a new peak which eluted slightly earlier than methionine. These data are consistent with the non-enzymatic glycation of lens proteins by either ascorbic acid or an oxidation product of ascorbic acid via a Maillard-type reaction.

The precipitation and cross-linking of lens crystallins by ascorbic acid.

Bovine lens beta-crystallin was incubated with increasing concentrations of sugars and sugar derivatives for a period of 2 weeks in the dark at 37 degrees C. Marked protein precipitation and a browning reaction was observed with both ascorbic acid (ASA) and dehydroascorbic acid (DHA), but little or no reaction was seen with several other sugars and sugar analogs. Similar incubations were carried out with 20 mM ASA, 20 mM DHA and 20 mM glucose, but with increasing amounts of the individual crystallins. Glucose was capable of precipitating gamma-crystallin in the presence of air, but this reaction was decreased if dithiothreitol and a chelating agent were added prior to incubation. ASA and DHA produced precipitation and browning with gamma- and beta-crystallin, but not with alpha-crystallin or lens soluble proteins. Similar reactivities were observed both in air and under reducing conditions. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of these reaction mixtures showed little or no cross-linking with any of the lens proteins by glucose. ASA and DHA caused detectable dimer formation with gamma-crystallin, but produced the formation of dimers as well as highly polymerized proteins at the top of the gel with all the other crystallins and with lens soluble proteins. A time-course experiment with alpha-crystallin in the presence of air showed no cross-linking with 100 mM glucose over a 6-week period; however, 10 mM ASA caused definite cross-linking at 2 weeks, and at 6 weeks a dark smear of protein was visible throughout the gel. ASA was still capable of inducing cross-linking under low oxygen conditions but the protein smearing was markedly diminished. Further, the cross-linking pattern was similar to that seen in the water-insoluble fraction from older human lenses and cataracts. This reaction may be significant in vivo because cross-linking was observed under low-oxygen conditions with as little as 2 mM ASA, which is the level of ASA normally present in human lenses.

Glutathione inhibits the glycation and crosslinking of lens proteins by ascorbic acid

The incubation of crude extracts of bovine lens with 20 mM ascorbic acid leads to the formation of covalent adducts even in the presence of saturating levels of a metal chelator. When dialysed lens extracts were used both ASA-protein adducts and highly crosslinked lens proteins were observed which are similar to those found in the water insoluble fraction from cataractous lenses. Both adduct formation and protein crosslinking, however, were markedly inhibited if undialysed lens extracts were used or if increasing concentrations of glutathione were added to the incubation mixture. Similar inhibition was seen with cysteine, dithiothreitol and sodium bisulfite, but little effect was observed with the glutathione analog ophthalmic acid or with free radical quenchers. Glutathione was readily oxidized during the incubation and no oxidation of ascorbic acid was observed until all the reduced glutathione was exhausted. No loss of ascorbic acid and no protein crosslinking were observed when oxygen was completely removed from the reaction mixture. These data strongly suggest that the glycating species was an oxidized form of ascorbic acid. Ascorbic acid solutions displayed a rapid oxidation in vitro, which was decreased 80-fold upon the addition of 1 mM chelator and was completely inhibited by both glutathione and chelator. A rapid decrease in the level of dissolved oxygen was seen in the presence of ascorbic acid or ascorbic acid and glutathione, but not with glutathione alone. These data argue that glutathione inhibits glycation by rapidly reducing dehydroascorbic acid back to ascorbic acid, which is not active in protein glycation

ASCORBIC ACID GLYCATION OF LENS PROTEINS PRODUCES UVA SENSITIZERS SIMILAR TO THOSE IN HUMAN LENS

‐Soluble calf lens proteins were extensively glycated during a 4 week incubation with ascorbic acid in the presence of oxygen. Amino acid analysis of the dialyzed proteins removed at weekly intervals showed an increasing loss of lysine, arginine and histidine, consistent with the extensive protein cross‐linking observed. Irradiation of the dialyzed samples with UVA light (1.0 kJ/cm2 total illumination through a 338 nm cutoff filter) caused an increasing loss of tryptophan, an additional loss of histidine and the production of micromolar concentrations of hydrogen peroxide. No alteration in amino acid content and no photolytic effects were seen in proteins incubated without ascorbic acid or in proteins incubated with glucose for 4 weeks. The rate of hydrogen peroxide formation was linear with each glycated sample with a maximum production of 25 nmol/mg protein illuminated. The possibility that the sensitizer activity was due to an ascorbate‐induced oxidation of tryptophan was eliminated by the presence of a heavy metal ion chelator during the incubation and by showing equivalent effects with ascorbate‐incubated ribonuclease A, which is devoid of tryptophan. The ascorbate‐incubated samples displayed increasing absorbance at wavelengths above 300 nm and increasing fluorescence (340/430) as glycation proceeded. The spectra of the 4 week glycated proteins were identical to those obtained with a solubilized water‐insoluble fraction from human lens, which is known to have UVA sensitizer activity.

UVA PHOTOLYSIS USING THE PROTEIN-BOUND SENSITIZERS PRESENT IN HUMAN LENS

Abstract—This research was undertaken to demonstrate that the protein‐bound chromophores in aged human lens can act as sensitizers for protein damage by UVA light. The water‐insoluble (WI) proteins from pooled human and bovine lenses were solubilized by sonication in water and illuminated with UV light similar in output to that transmitted by the cornea. Analysis of the irradiated proteins showed a linear decrease in sulfiydryl groups with a 30% loss after 2 h. No loss was seen when native a‐crystallin was irradiated under the same conditions. A 25% loss of histidine residues was also observed with the human lens WI fraction, and sodium dodecyl sulfate polyacrylamide gels indicated considerable protein cross‐linking. Similar photodamage was seen with a WI fraction from old bovine lenses. While the data show the presence of UVA sensitizers, some histidine destruction and protein cross‐linking were also obtained with a‐crystallin and with lysozyme, which argue that part of the histidine loss in the human WISS was likely due to tryptophan acting as a sensitizer.

Studies on the solubilization of the water-insoluble fraction from human lens and cataract

Studies were carried out comparing the ability of urea extraction and sonication to solubilize the water-insoluble (WI) protein fraction from human lens tissue. Sonication and urea extraction were able to solubilize greater than 80% of the insoluble protein whether whole lenses or lens nuclei were used. This was true for normal lens and +1 cataracts; however, only 60% solubilization was obtained with the WI fraction from more advanced cataracts. Equal aliquots of a WI fraction from both pooled normal and pooled cataract lens nuclei were solubilized with and without reducing agents. The addition of dithiothreitol (DTT) had no significant effect on solubilization of the normal lens WI fraction. DTT did increase the protein solubilized from the cataract WI fraction by 30% with urea extraction; however, no increase was seen with sonication. When sodium borohydride was used as the reducing agent, essentially the same results were obtained. The solubilized protein populations were identical by SDS-PAGE and amino acid analysis. The addition of reducing agents had no effect on the amino acid content of the solubilized proteins with the single exception of lysine. This amino acid was markedly decreased in the proteins extracted in the presence of 40 mM sodium borohydride, but not with DTT. These data suggest that the borohydride not only increased the amount of protein solubilized, but likely also stabilized glycated lysine residues during the acid hydrolysis. Therefore, sonication readily provides a soluble preparation of the WI proteins from normal and cataract lens nuclei without the need for denaturing agents, however, disulfide-linked and lysine modified crystallins were best solubilized with urea.

Solubilization of the lens water-insoluble fraction by sonication

A method is reported whereby the solubilization of the bulk of the lens water-insoluble fraction is accomplished by a short sonication of the suspended proteins in low salt buffers. This procedure solubilized greater than 90% of a bovine lens water-insoluble fraction and 80% of the normal human lens water-insoluble fraction. Decreased protein was solubilized from cataractous lenses, but in every case sonication was at least equivalent to extraction with 6.0 M urea. Fractionation of the solubilized proteins by Agarose A-1.5 m gel filtration chromatography showed native molecular weights for bovine lens, but only partial disaggregation with human lens extracts. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) comparison of the proteins solubilized by sonication and 6.0 M urea extraction showed no major differences except that sonication solubilized more of the highly cross-linked protein which remained at the top of the gel.

Isolation and characterization of a new advanced glycation endproduct of dehydroascorbic acid and lysine

Proteins are subject of posttranslational modification by sugars and their degradation products in vivo. The process is often referred as glycation. L-Dehydroascorbic acid (DHA), an oxidation product of L-ascorbic acid (vitamin C), is known as a potent glycation agent. A new product of modification of lysine epsilon -amino group by DHA was discovered as a result of the interaction between Boc-Lys and dehydroascorbic acid. The chromatographic and spectral analyses revealed that the structure of the product was 1-(5-ammonio-5-carboxypentyl)-3-oxido-4-(hydroxymethyl)pyridinium. The same compound was isolated from DHA modified calf lens protein after hydrolysis and chromatographic separation. The study confirmed that L-erythrulose is an important intermediate of modification of proteins by DHA. The structure of the reported product and in vitro experiments suggested that L-erythrulose could further transform to L-threose, L-erythrose and glycolaldehyde under conditions similar to physiological. The present study revealed that the modification of epsilon -amino groups of lysine residues by DHA is a complex process and could involve a number of reactive carbonyl species.

Studies on the nature of the water-insoluble fraction from aged bovine lenses

The water-insoluble fraction from mature bovine lens was solubilized to the same extent either by extraction with 6.0 M urea, by sonication of the suspended proteins or by a brief adjustment of the pH to 3.0 or 11.0. Sonication gave soluble protein levels of 50 mg ml-1 or greater with water or dilute buffers, but the presence of salt markedly diminished the solubility of the sonicated proteins. The sonicated proteins remained soluble upon storage at 5 degrees C, but were readily precipitated by either freezing or by the addition of salt. These re-precipitated proteins were once again insoluble when suspended in dilute aqueous buffers. Water-soluble alpha-crystallin at the same concentrations was unaffected by either high salt or freezing. The sonication-solubilized proteins were shown to be similar in aggregate size and polypeptide composition to the water-soluble HMW fraction isolated from the same lenses. An [125I]-labeled soluble HMW fraction was precipitated to the same extent as [125I]-labeled sonication-solubilized proteins upon freezing. The distribution of HMW aggregated protein between water-soluble aggregates and the water-insoluble fraction was unaltered by the presence of either dithiothreitol (DTT) or high levels of salt during the homogenization. The presence of either [125I]-labeled water-soluble HMW aggregates or [125I]-labeled water-insoluble sonicate supernatant during lens homogenization did not result in a significant incorporation of radioactivity into the water-insoluble fraction. These data argue that the water-insoluble fraction represents coalesced HMW aggregates which had already formed in the lens prior to homogenization. When the sonication-solubilized fraction was disaggregated in 6.0 M urea and then reaggregated by urea removal, the proteins no longer precipitated on freezing, and 85-90% of the protein eluted in the region of alpha-crystallin from an Agarose A-5m column. Only 3-6% of the original protein remained as a void volume peak, and was composed almost exclusively of highly crosslinked proteins. The limited solubility of the HMW proteins may therefore reflect the aggregate state of the alpha-crystallin rather than an inherent insolubility of the subunits.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of the elastase inhibitor properties of α-crystallin and the water-insoluble fraction from bovine lens

Alpha-crystallin exhibits variable inhibition of several members of the chymotrypsin family of proteinases. Complete inhibition of elastase was obtained by the addition of either alpha-crystallin or a sonicated preparation of the water-insoluble fraction from bovine lens. Little or no inhibition was seen, however, with either beta-crystallin or bovine serum albumin under the same conditions. Complete binding of elastase was demonstrated by Sephadex G-100 gel filtration chromatography, and a direct correlation between binding and inhibition was obtained. This observation permitted us to do a Scatchard analysis of the inhibition data. Scatchard plots for the binding of elastase gave a biphasic response suggesting two separate binding sites. These sites had Kd values of 15 and 40 nM for alpha-crystallin and 6 and 42 nM for the bovine water-insoluble fraction. Similarly, a Dixon plot exhibited a Ki value of 3 nM and was consistent with non-competitive inhibition. One mole of alpha-crystallin (8 x 10(5) Da), or an equivalent amount of water-insoluble protein, bound from 13 to 19 mol of elastase which were about equally divided between the higher and lower affinity sites. Saturation studies confirmed 20 and 16 elastase binding sites per 8 x 10(5) Da for alpha-crystallin and water-insoluble protein, respectively. DFP-elastase was capable of binding to alpha-crystallin suggesting that a proteolytic cleavage was not required for complex formation. Stability measurements showed a linear return to 60% of the original activity over a 30-min period. Therefore, the interaction between elastase and alpha-crystallin resembles that of a heterologous protease:inhibitor complex in both binding and stability.