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 non-oxidative degradation of ascorbic acid at physiological conditions.

The degradation of L-ascorbate (AsA) and its primary oxidation products, L-dehydroascorbate (DHA) and 2,3-L-diketogulonate (2, 3-DKG) were studied under physiological conditions. Analysis determined that L-erythrulose (ERU) and oxalate were the primary degradation products of ASA regardless of which compound was used as the starting material. The identification of ERU was determined by proton decoupled (13)C-nuclear magnetic resonance spectroscopy, and was quantified by high performance liquid chromatography, and enzymatic analysis. The molar yield of ERU from 2,3-DKG at pH 7.0 37 degrees C and limiting O(2)97%. This novel ketose product of AsA degradation, was additionally qualitatively identified by gas-liquid chromatography, and by thin layer chromatography. ERU is an extremely reactive ketose, which rapidly glycates and crosslinks proteins, and therefore may mediate the AsA-dependent modification of protein (ascorbylation) seen in vitro, and also proposed to occur in vivo in human lens during diabetic and age-onset cataract formation.

Inhibitors of advanced glycation end product-associated protein cross-linking.

The reaction of lens proteins with sugars over time results in the formation of protein-bound advanced glycation end products (AGEs). The most damaging element of AGE formation may be the synthesis of protein-protein cross-links in long-lived proteins, such as collagen or lens crystallins. A quantitative cross-linking assay, involving the sugar-dependent incorporation of [U-(14)C]lysine into protein, was employed to determine the efficacy of a variety of potential cross-linking inhibitors. Reaction mixtures contained 5.0 mM L-threose, 2.5 microCi [(14)C]lysine (1.0 mCi/mmole), 5.0 mg/ml bovine lens proteins, 0-10 mM inhibitor and 1.0 mM DTPA in 100 mM phosphate buffer, pH 7.0. Of 17 potential inhibitors tested, 11 showed 50% inhibition or less at 10 mM. The dicarbonyl-reactive compounds 2-aminoguanidine, semicarbazide and o-phenylenediamine inhibited 50% at 2.0 mM, whereas 10 mM dimethylguanidine had no effect. Several amino acids failed to compete effectively with [(14)C]lysine in the cross-linking assay; however, cysteine inhibited 50% at 1.0 mM. This was likely due to the sulfhydryl group of cysteine, because 3-mercaptopropionic acid and reduced glutathione exhibited similar activity. Sodium metabisulfite had the highest activity, inhibiting 50% at only 0.1-0.2 mM. Protein dimer formation, as determined by SDS-PAGE, was inhibited in a quantitatively similar manner. The dicarbonyl-reactive inhibitors and the sulfur-containing compounds produced similar inhibition curves for [(14)C]lysine incorporation over a 3 week assay with 250 mM glucose. A much lesser effect was observed on either the incorporation of [(14)C]glucose, or on fluorophore formation (360/420 nm), suggesting that non-cross-link fluorophores were also formed. The inhibitor data were consistent with cross-linking by a dicarbonyl intermediate. This was supported by the fact that the inhibitors were uniformly less effective when the 5.0 mM threose was replaced by either 3.0 mM 3-deoxythreosone or 3.0 mM threosone.

The binding and inhibition of trypsin by alpha-crystallin.

One of the major lens-structural proteins, alpha-crystallin, is a multimeric protein containing 40 subunits of approx. 20 kDa each. There are two subunit types with distinct but similar structures. This protein was capable of inhibiting trypsin, chymotrypsin and elastase, but had no effect on thrombin or kallikrein. Complete inhibition was not observed, but rather plateau levels of inhibition were obtained in each case. Maximum inhibition was observed at a ratio of 1 mol of alpha-crystallin for every 9-10 mol of trypsin. alpha-Crystallin also inhibited the labeling of the active site of trypsin by [3H]diisopropyl fluorophosphate (DFP). Greater than 90% inhibition of DFP labeling was observed at a ratio of 1 mol of alpha-crystallin for every 7-8 mol of trypsin. Both trypsin and [3H]DFP-labeled trypsin formed a complex with alpha-crystallin, as demonstrated by gel-filtration chromatography. The active site of trypsin when bound to alpha-crystallin was still capable of reacting with p-nitrophenyl p-guanidobenzoate and soybean trypsin inhibitor, but was inaccessible to alpha 1-antitrypsin. These data suggest that alpha-crystallin acts as a multivalent modified inhibitor which is consistent with the proposed quaternary structure of alpha-crystallin.

Glycation of lens proteins by the oxidation products of ascorbic acid.

Bovine lens water-soluble proteins were incubated with [I-14C]ascorbic acid (ASA) for 6 days, and the incorporation into protein was measured at daily intervals. Aliquots were also withdrawn to determine the distribution of label among the various ASA oxidation products. A linear incorporation into protein was observed in the presence of NaCNBH3, however, little or no incorporation was seen in its absence. TLC analysis showed a complete loss of ASA by day 3, whereas both dehydroascorbate (DHA) and diketogulonic acid (DKG) remained constant for 6 days, consistent with the linear incorporation into protein. The amino acid composition of the proteins glycated in the presence of NaCNBH3 was identical to controls except for a 70% reduction in lysine residues and a corresponding increase in an unknown product which eluted slightly earlier than methionine. In the absence of NaCNBH3 lysine decreased linearly to 20% with an additional decrease in arginine and histidine at later times concurrent with protein crosslinking. DHA and DKG were prepared and incubated directly with lens proteins for an 8 day period. Both compounds glycated lens protein as evidenced by an increased binding to a boronate affinity column. SDS-PAGE showed that both compounds were also capable of causing protein crosslinking. DHA is apparently capable of reacting directly with protein since glycation was observed with the ASA analog, reductic acid, which can be oxidized to dehydroreductic acid, but which cannot be hydrolyzed to an open chain structure. DHA also produced a lysine adduct which was not obtained with DKG, supporting the idea that both species have glycating ability.

Similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid-modified calf lens proteins: evidence for ascorbic acid glycation during cataract formation.

Chromatographic evidence supporting the similarity of the yellow chromophores isolated from aged human and brunescent cataract lenses and calf lens proteins ascorbylated in vitro is presented. The water-insoluble fraction from early stage brunescent cataract lenses was solubilized by sonication (WISS) and digested with a battery of proteolytic enzymes under argon to prevent oxidation. Also, calf lens proteins were incubated with ascorbic acid for 4 weeks in air and submitted to the same digestion. The percent hydrolysis of the proteins to amino acids was approximately 90% in every case. The content of yellow chromophores was 90, 130 and 250 A(330) units/g protein for normal human WISS, cataract WISS and ascorbate-modified bovine lens proteins respectively. Aliquots equivalent to 2.0 g of digested protein were subjected to size-exclusion chromatography on a Bio-Gel P-2 column. Six peaks were obtained for both preparations and pooled. Side by side thin-layer chromatography (TLC) of each peak showed very similar R(f) values for the long wavelength-absorbing fluorophores. Glycation with [U-(14)C]ascorbic acid, followed by digestion and Bio-Gel P-2 chromatography, showed that the incorporated radioactivity co-eluted with the A(330)-absorbing peaks, and that most of the fluorescent bands were labeled after TLC. Peaks 2 and 3 from the P-2 were further fractionated by preparative Prodigy C-18 reversed-phase high-performance liquid chromatography. Two major A(330)-absorbing peaks were seen in peak 2 isolated from human cataract lenses and 5 peaks in fraction 3, all of which eluted at the same retention times as those from ascorbic acid glycated calf lens proteins. HPLC fractionation of P-2 peaks 4, 5 and 6 showed many A(330)-absorbing peaks from the cataract WISS, only some of which were identical to the asorbylated proteins. The major fluorophores, however, were present in both preparations. These data provide new evidence to support the hypothesis that the yellow chromophores in brunescent lenses represent advanced glycation endproducts (AGEs) probably due to ascorbic acid glycation in vivo.

The extent of N epsilon-(carboxymethyl)lysine formation in lens proteins and polylysine by the autoxidation products of ascorbic acid.

The autoxidation of ascorbic acid (ASA) leads to the formation of compounds which are capable of glycating and crosslinking proteins in vitro. When the soluble crystallins from bovine lens were incubated with ASA in the presence of sodium cyanoborohydride, a single major adduct was observed, whose appearance correlated with the loss of lysine. When polylysine was reacted with equivalent amounts of ASA under the same conditions, this product represented half of the total lysine content after four weeks of incubation at 37 degrees C. This adduct was isolated and identified as N epsilon-(carboxymethyl)lysine (CML) by TLC, GC/MS and amino acid analysis. Several oxidation products of ASA were each reacted with polylysine in the presence of sodium cyanoborohydride to identify the reactive species. CML was the major adduct formed with either ASA and dehydroascorbic acid (DHA). Markedly diminished amounts were seen with L-2,3-diketogulonic acid (DKG), and L-threose, while no CML was formed with L-threo-pentos-2-ulose (L-xylosone). In the absence of sodium cyanoborohydride the yield of CML was similar with each of the ASA autoxidation products and required oxygen. Reactions with [1-14C]ASA gave rise to [14C]CML, but only with NaCNBH3 present. At least two routes of CML formation appear to be operating depending upon whether NaCNBH3 is present to reduce the putative Schiff base formed between lysine and DHA.

Site-specific glycation of lens crystallins by ascorbic acid.

The oxidation of ascorbic acid leads to the formation of several compounds which are capable of reacting with protein amino groups via a Maillard reaction. Radioactivity from [1-14C]ascorbic acid was linearly incorporated into lens crystallins over a 10 day period in the presence of NaCNBH3. This rate of incorporation was 6-7-fold more rapid than that obtained with [14C]glucose under the same conditions. SDS-PAGE showed a linear incorporation into all the crystallin subunits. [1-14C]Ascorbic acid-label led alpha-crystallin was separated into its component A and B subunits, and each was digested with chymotrypsin. HPLC peptide analysis showed a differential labelling of the various lysine residues. Analysis of the peptides by mass spectrometry allowed the identification of the sites and the extent of modification. These values ranged from 6% for Lys-78 to 36% for Lys-11 in the A subunit and from 5% for Lys-82 to an average of 38% for the peptide containing Lys-166, Lys-174 and Lys-175 in the B subunit. Amino acid analysis demonstrated a single modification reaction producing N epsilon-(carboxymethyl)lysine. This agreed with the mass increase of 58 observed for each modified peptide.

Suppression of pentosidine formation in galactosemic rat lens by an inhibitor of aldose reductase.

Recent work from our laboratory revealed a correlation between the degree of protein pigmentation in human cataractous lens and the advanced Maillard reaction as reflected by pentosidine formation. Although the data suggested a role for ascorbate in pentosidine formation in senile cataractous lenses, elevated pentosidine levels in diabetic cataracts suggested that glucosylation may be involved directly in pentosidine biosynthesis. To clarify this issue, we quantified pentosidine in lenses from rats with experimental galactosemia with and without aldose reductase inhibitor treatment. At 12 months, pentosidine-like fluorescence (335/385 nm) was three to six times higher (P < 0.0001) in water soluble and insoluble crystallins of galactosemic compared with nongalactosemic rats. Actual pentosidine levels increased shortly after onset of galactosemia. Contents in water-insoluble crystallins were 6.32 +/- 2.2 and 1.40 +/- 0.66 pmol/mg protein in galactosemic and control lenses, respectively (P < 0.001). Fluorescence and pentosidine were suppressed to almost control levels upon treatment with sorbinil. Incubation experiments showed that pentosidine could form slowly from galactose, but much more rapidly from ascorbate and its oxidation products. Its formation could be inhibited partly by both reduced and oxidized glutathione or epsilon-aminocaproic acid. The requirement of oxygen for pentosidine formation suggests that oxidative stress associated with glutathione depletion and ascorbate oxidation are plausible mechanisms for rapid pentosidine formation upon onset of galactosemia. In contrast, Maillard reaction by glycoxidation products may account for the sustained increase in pentosidine. Both these events may be linked to the newly recognized pseudohypoxic state of cells exposed to high sugar concentrations.

Phototransformations of advanced glycation end products in the human eye lens due to ultraviolet A light irradiation.

Previous studies from this laboratory have shown that ultraviolet A (UVA) light can bleach the yellow advanced glycation end products (AGEs) of aged and cataractous human lenses. The AGEs OP-lysine and argpyrimidine are two UVA-absorbing posttranslational modifications that are abundant in the eye lens. The purpose of this study was to outline the changes in these two AGEs due to UVA irradiation. The changes of OP-lysine, OP-phenethylamine (a phenethylamine analogue of OP-lysine), and argpyrimidine due to irradiation with UVA light in the presence or absence of air and ascorbic acid were followed by different spectral methods. Aged human lenses were similarly irradiated in artificial aqueous humor. The amounts of OP-lysine in the irradiated lenses and in the corresponding dark controls were determined by HPLC. Both OP-lysine and argpyrimidine decreased 20% when irradiated with UVA light in the absence of ascorbic acid. Under the same conditions, OP-lysine was bleached 80% in the presence of ascorbic acid during irradiation experiments. In contrast, argpyrimidine UVA light bleaching was not affected by the presence of ascorbic acid. Interestingly the major product of OP-phenethylamine after UVA irradiation in the presence of ascorbic acid was phenethylamine, which indicates that the entire heterocycle of this AGE was cleaved and the initial amino group was restored. Some AGEs in the human eye lens can be transformed by UVA light.

Glycation mediated crosslinking between alpha-crystallin and MP26 in intact lens membranes.

With advancing age, progressive crosslinking occurs between lens crystallin proteins and other lenticular components. This crosslinking may be involved in the development of senile cataracts. Experiments were conducted to determine whether non-enzymatic glycation could be involved in the crosslinking between lens alpha-crystallin and MP26, an abundant lens fiber cell membrane intrinsic protein. In vitro crosslinking of alpha-crystallin and MP26 of bovine lens membranes was observed in presence of two degradation products of ascorbic acid (ASA), dehydroascorbic acid (DHA) and threose. Alkali-washed bovine lens membranes, isolated after glycation with DHA and threose, contained both alpha-crystallin and MP26, as determined by immunoblot and double immunocytochemical labeling studies. In contrast, membranes incubated without these glycating compounds contained only MP26. SDS-PAGE analysis of [125I] alpha-crystallin incubated with lens membranes in the presence of threose showed a higher amount of radioactivity in high molecular weight aggregates than in the aggregates produced when alpha-crystallin and threose were incubated without membranes. A slot-blot immunoassay of alkali-washed human lens membranes showed a higher amount of covalently bound alpha-crystallin in aged, cataractous or diabetic lens membranes than was present in lens membranes from young normal donors. Based on the in vitro results, we hypothesize that non-enzymatic glycation is one of the vivo mechanisms in the crosslinking of alpha-crystallin to lens membrane proteins, such as MP26. This crosslinking may contribute significantly to the development of age-related and diabetic cataracts.

Ascorbic acid and glucose oxidation by ultraviolet A-generated oxygen free radicals.

PURPOSE: To determine the relative rate of oxidation of ascorbic acid (ASA) and glucose under conditions used for glycation reactions in vitro and by ultraviolet A (UVA)-generated oxygen free radicals using human lens sensitizers. METHODS: ASA and [14C]glucose were incubated in 0.1 M phosphate buffer, and the rate of oxidation was determined by absorbance at 265 nm and by thin-layer chromatography, respectively. Oxidation also was measured during the UVA irradiation of 2 mg/ml solutions of human lens water-insoluble proteins. The role of individual reactive oxygen species was determined by the protective effects of superoxide dismutase, catalase, and sodium azide. RESULTS: ASA was oxidized rapidly in 0.1 M phosphate buffer. This loss was prevented by the addition of a metal chelator, by previous chelex resin treatment of the buffer, or by the addition of lens proteins. Glucose was not oxidized under any of the above conditions. UVA irradiation with 2 mg/ml human lens protein as sensitizer oxidized 1 mM ASA after several hours but oxidized, at most, only 2 microM glucose even after 8 hours of irradiation. Superoxide anion was responsible for 24%, and singlet oxygen for 40%, of the ASA oxidized. UVA-generated H2O2 caused little or no oxidation of ASA. H2O2 did accelerate the oxidation of ASA in phosphate buffer, but this was almost completely prevented by the addition of either a chelating agent or lens proteins. CONCLUSIONS: The conditions used for glycation reactions in vitro rapidly oxidized ASA, but not glucose. The UVA-dependent generation of oxygen free radicals also oxidized ASA at a 10(3) faster rate than glucose. Superoxide anion and singlet oxygen were identified as the principal oxidants of ASA in this process. These data argue that ASA may be the primary glycating agent in aging normal lenses.

The effects of growth factors on tRNALys modification.

The tRNALys population from tissue culture cells contains several isoaccepting species which are not present in the tRNALys population from tissue sources. These isoacceptors were isolated from mouse LM cells and tested for their coding properties in ribosomal binding assays and their ability to incorporate lysine into protein in a reticulocyte lysate. tRNALys5A and tRNALys6 eluted in the area of tRNALys5. All three species coded preferentially for AAA and bound with equal efficiency. tRNALys1, tRNALys3, tRNALys4, and tRNALys6 all transferred lysine into protein at a slower rate than tRNALys2 and tRNALys5, which are the native species. Several purified growth factors were tested for their ability to affect the levels of tRNALys4, a tRNA possibly associated with cell division. When Balb/c 3T3 cells were grown in medium containing plasma instead of serum, there was a decrease in tRNALys2, tRNALys3, tRNALys4 and an increase in tRNALys5 and tRNALys6. The addition of either FGF or PDGF returned the tRNALys profile to normal. The extent of the tRNALys changes depended upon the concentration of growth factor added. FGF was able to cause a 35% decrease in the tRNALys5 peak with a corresponding increase in tRNALys2 within 1 h of the addition of the factor. These data suggest that competence factors have the ability to stimulate the modification of specific tRNALys isoacceptors.

Perturbation of the mitochondrial lysine tRNA population by virus-induced transformation or stress of mammalian cells: functional properties and nucleotide sequence of a mitochondrially associated lysine tRNA.

Eleven isoaccepting lysine tRNAs from mammalian sources are demonstrable by RPC-5 chromatography and polyacrylamide gel electrophoresis. The appearance and amounts of these isoacceptors varies with the source and growth state of cells. One isoacceptor, tRNALys6, observed in preparations of tRNA from some virus-transformed cells in culture, has been characterized by determining functional properties, cellular location, and its nucleotide sequence. tRNALys6 responds primarily to the lysine codon AAA, but it is not used efficiently in a wheat germ translational system in vitro. Compared with lysine isoacceptors 1, 2, 4, 5a, and 5, [3H]lysine appears in vivo in tRNALys6 with a delay of about 3 h. This delay may in part be a result of a less functional tRNA, but a compartmented state of tRNALys6 also appears to be important. tRNALys6 is associated with mitoplasts prepared from KA31 fibroblasts. The nucleotide sequence of tRNALys6 was determined by rapid postlabeling procedures involving limited hydrolysis in formamide, 32P-labeling of 5' ends of fragments with polynucleotide kinase, separation of the nested set of fragments in polyacrylamide denaturing gels, release of 5'-labeled nucleotides with RNase T2, and identification of the released nucleotides by chromatography on PEI cellulose. Confirmation of the positions of major nucleotides was done by using limited digestions by RNases of tRNALys6 labeled with 32P on the 3' terminus in a gel readout procedure. The nucleotide sequence of tRNALys6 differs from that of cytoplasmic lysine tRNAs and mammalian mitochondrial lysine tRNAs. It contains U*, an unidentified modified uridine occurring in the anticodon of some mitochondrial tRNAs. tRNALys6 appears to occur in very limited amounts, or not at all, in most cells unless stressed, but when present it is associated with mitochondria, although it is probably coded in the nucleus.

Quantitation of the reactive oxygen species generated by the UVA irradiation of ascorbic acid-glycated lens proteins.

The oxidation products of ascorbic acid rapidly glycate proteins and produce protein-bound, advanced glycation endproducts. These endproducts can absorb UVA light and cause the photolytic oxidation of proteins (Ortwerth, Linetsky and Olesen, Photochem. Photobiol. 62, 454-463, 1995), which is mediated by the formation of reactive oxygen species. A dialyzed preparation of calf lens proteins, which had been incubated for 4 weeks with 20 mM ascorbic acid in air, was irradiated for 1 h with 200 mW/cm2 of absorbed UVA light (gamma > 338 nm), and the concentration of individual oxygen free radicals was measured. Superoxide anion attained a level of 76 microM as determined by the superoxide dismutase (SOD)-dependent increase in hydrogen peroxide formation and of 52 microM by the SOD-inhibitable reduction of cytochrome c. Hydrogen peroxide formation increased linearly to 81 microM after 1 h. Neither superoxide anion nor hydrogen peroxide, however, could account for the UVA photolysis of Trp and His seen in this system. Singlet oxygen levels approached 1.0 mM as measured by the oxidation of histidine, which was consistent with singlet oxygen measurements by the bleaching of N,N-dimethyl-4-nitrosoaniline. High concentrations of sodium azide, a known singlet oxygen quencher, inhibited the photolytic destruction of both His and Trp. Little or no protein damage could be ascribed to hydroxyl radical based upon quenching experiments with added mannitol. Therefore, superoxide anion and H2O2 were generated by the UVA irradiation of ascorbate advanced glycation endproducts, however, the major reactive oxygen species formed was singlet oxygen.

UVA photolysis using the protein-bound sensitizers present in human lens.

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 sulfhydryl groups with a 30% loss after 2 h. No loss was seen when native alpha-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 alpha-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. A preparation of human WI proteins was irradiated with a total of 200 J/cm2 of absorbed light at 10 nm intervals from 290 to 400 nm. Photodamage of cysteine SH groups (35%) and methionine (28%) was maximum at 330 nm and diminished linearly at longer wavelengths. The major loss of tryptophan (80%) occurred at 290 nm, but destruction was observed throughout the UVA range. Tyrosine was 35% destroyed at 290 nm but decreased sharply to only 5% at 330 nm.(ABSTRACT TRUNCATED AT 250 WORDS)

The generation of hydrogen peroxide by the UVA irradiation of human lens proteins.

The water-insoluble proteins from aged human lens are known to contain protein-bound chromophores that act as UVA senisitizers. The irradiation of a sonication-solubilized, water-insoluble fraction from human lenses (55-75 years) with UVA light (1.5 kJ/cm2, gamma > 338nm) caused an oxygen-dependent photolysis of tryptophan, not seen when either alpha-crystallin or lysozyme were irradiated. The suggested requirement for active oxygen species was consistent with a linear increase in hydrogen peroxide formation, which was also observed. A final concentration of 55 microM H2O2 was attained, with no H2O2 being detected in either dark-incubated controls or in irradiated samples of native proteins. The UVA-dependent H2O2 formation was increased 50% by superoxide dismutase (SOD) and abolished by catalase, arguing for the initial generation of superoxide anion. A linear photolysis of histidine and tryptophan was also seen; however, the addition of SOD or SOD and catalase had no effect on the photolytic destruction of either amino acid. Superoxide dismutase increased the oxidation of protein SH groups implicating H2O2, but SOD and catalase caused a decrease in SH oxidation only at later time periods. The direct addition of H2O2 to a water-insoluble sonicate supernatant fraction caused only a slight oxidation of SH groups, but this was increased four- to eight-fold when the protein was denatured in 4.0 M guanidine hydrochloride. Overall, the data suggest a UVA-dependent oxidation of protein SH groups via H2O2 generated within the large protein aggregates of the water-insoluble fraction. These data also provide a mechanism for oxidation of the sulfur-containing amino acids in vivo--a process that is known to accompany the formation of age-onset cataracts.

Quantitation of the singlet oxygen produced by UVA irradiation of human lens proteins.

Ultraviolet irradiation of aged human lens proteins in vitro causes extensive photolytic damage of His and Trp residues. Protection by sodium azide argues for a process mediated by singlet oxygen (1O2). In the work described here, the synthesis of 1O2 was measured by the bleaching of N,N-dimethyl-4-nitrosoaniline (RNO), the oxidation of added histidine and the oxidation of furfuryl alcohol. To obtain a more accurate value for 1O2 generation, a known quantity of 1O2 was generated by the thermal dissociation of 3-(4-methyl-naphthyl)propionic acid endoperoxide, and the efficiency of each assay method to report on the 1O2 generated was determined. The values obtained were 0.003 mol of RNO bleached/mol of 1O2 generated, 0.55 mol of furfuryl alcohol oxidized/mol 1O2 and 0.5 mol of His oxidized/mol 1O2 generated. Irradiation of the human lens proteins with UVA light produced from 2.1 to 2.4 mM of 1O2 by RNO bleaching, 2.6-2.8 mM 1O2 by furfuryl alcohol oxidation and up to 1.9 mM of 1O2 by histidine oxidation during a 1 h irradiation period. The average value (2.2 mM of 1O2) corresponds to the theoretical production of 30 nmol of singlet oxygen at UVA light intensities equivalent to a 1 h exposure to sunlight at noon in the northern hemisphere.

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. The incubation of lens proteins with dehydroascorbic acid or L-threose, but not fructose, produced equivalent glycation, protein crosslinking and sensitizer activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The relative UV sensitizer activity of purified advanced glycation endproducts.

The oxidation products of ascorbic acid react with lens proteins to form advanced glycation endproducts (AGE) that are capable of generating reactive oxygen species when irradiated with UVA light. L-Threose, the most active of these oxidation products, was reacted with N-acetyl lysine and six AGE peaks were isolated by RP-HPLC. Each peak exhibited fluorescence and generated superoxide anion and singlet oxygen in response to UV light. Solutions of these AGE peaks (50 micrograms/mL) generated 5-10 nmol/mL of superoxide anion during a 30 min irradiation. This activity was 100-fold less than the superoxide anion generated by kynurenic acid and 400-fold less than riboflavin. Ultraviolet irradiation generated from 1.2 to 2.7 mumol/mL of singlet oxygen with the purified threose AGE compounds. This activity was similar to that seen with other purified AGE compounds (pentosidine, LM-1 and Ac-FTP) and with kynurenine and 3-OH kynurenine. This considerable singlet oxygen formation, however, was still 40-fold less than that obtained with kynurenic acid and 100-fold less than riboflavin under the same irradiation conditions. In spite of this lower sensitizer efficiency, the purified AGE generated 20-60-fold more singlet oxygen on a weight basis than either crude ascorbic acid glycated proteins or a preparation of water-insoluble proteins from aged normal human lenses. On a molar basis, therefore, AGE could account for the sensitizer activity in these protein preparations if they represented less than 1% of the total amino acids.