Hydration at the membrane protein-lipid interface.

Evidence has been found for the existence water at the protein-lipid hydrophobic interface of the membrane proteins, gramicidin and apocytochrome C, using two related fluorescence spectroscopic approaches. The first approach exploited the fact that the presence of water in the excited state solvent cage of a fluorophore increases the rate of decay. For 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5- hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-PC (DPH-PC), where the fluorophores are located in the hydrophobic core of the lipid bilayer, the introduction of gramicidin reduced the fluorescence lifetime, indicative of an increased presence of water in the bilayer. Since a high protein:lipid ratio was used, the fluorophores were forced to be adjacent to the protein hydrophobic surface, hence the presence of water in this region could be inferred. Cholesterol is known to reduce the water content of lipid bilayers and this effect was maintained at the protein-lipid interface with both gramicidin and apocytochrome C, again suggesting hydration in this region. The second approach was to use the fluorescence enhancement induced by exchanging deuterium oxide (D2O) for H2O. Both the fluorescence intensities of trimethylammonium-DPH, located in the lipid head group region, and of the gramicidin intrinsic tryptophans were greater in a D2O buffer compared with H2O, showing that the fluorophores were exposed to water in the bilayer at the protein-lipid interface. In the presence of cholesterol the fluorescence intensity ratio of D2O to H2O decreased, indicating a removal of water by the cholesterol, in keeping with the lifetime data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Accumulation of the hydroxyl free radical markers meta-, ortho-tyrosine and DOPA in cataractous lenses is accompanied by a lower protein and phenylalanine content of the water-soluble phase

Post-translational modifications of lens proteins play a crucial role in the formation of cataract during ageing. The aim of our study was to analyze protein composition of the cataractous lenses by electrophoretic and high-performance liquid chromatographic (HPLC) methods. Samples were obtained after extracapsular cataract surgery performed by phacoemulsification technique from cataract patients with type 2 diabetes mellitus (DM CAT, n = 22) and cataract patients without diabetes (non-DM CAT, n = 20), while non-diabetic non-cataractous lenses obtained from cadaver eyes served as controls (CONTR, n = 17). Lens fragments were derived from the surgical medium by centrifugation. Samples were homogenized in a buffered medium containing protease inhibitor. Soluble and insoluble protein fractions were separated by centrifugation. The electrophoretic studies were performed according to Laemmli on equal amounts of proteins and were followed by silver intensification. Oxidized amino acid and Phe content of the samples were also analyzed by HPLC following acid hydrolysis of proteins. Our results showed that soluble proteins represented a significantly lower portion of the total protein content in cataractous lenses in comparison with the control group (CONTR, 71.25%; non-DM CAT, 32.00%; DM CAT, 33.15%; p < 0.05 vs CONTR for both). Among the proteins, the crystallin-like proteins with low-molecular weight can be found both in the soluble and insoluble fractions, and high-molecular weight aggregates were found mainly in the total homogenates. In our HPLC analysis, oxidatively modified derivatives of phenylalanine were detected in cataractous samples. We found higher levels of m-Tyr, o-Tyr and DOPA in the total homogenates of cataractous samples compared to the supernatants. In all three groups, the median Phe/protein ratio of the total homogenates was also higher than that of the supernatants (total homogenates vs supernatants, in the CONTR group 1102 vs 633 micromol/g, in the DM CAT group 1187 vs 382 micromol/g and in the non-DM CAT group 967 vs 252 micromol/g; p < 0.05 for all). In our study we found that oxidized amino acids accumulate in cataractous lenses, regardless of the origin of the cataract. The accumulation of the oxidized amino acids probably results from oxidation of Phe residues of the non-water soluble lens proteins. We found the presence of high-molecular weight protein aggregates in cataractous total homogenates, and a decrease of protein concentration in the water-soluble phase of cataractous lenses. The oxidation of lens proteins and the oxidative modification of Phe residues in key positions may lead to an altered interaction between protein and water molecules and thus contribute to lens opacification.     

Accumulation of the hydroxyl free radical markers meta-, ortho-tyrosine and DOPA in cataractous lenses is accompanied by a lower protein and phenylalanine content of the water-soluble phase

Post-translational modifications of lens proteins play a crucial role in the formation of cataract during ageing. The aim of our study was to analyze protein composition of the cataractous lenses by electrophoretic and high-performance liquid chromatographic (HPLC) methods.

Samples were obtained after extracapsular cataract surgery performed by phacoemulsification technique from cataract patients with type 2 diabetes mellitus (DM CAT, n = 22) and cataract patients without diabetes (non-DM CAT, n = 20), while non-diabetic non-cataractous lenses obtained from cadaver eyes served as controls (CONTR, n = 17). Lens fragments were derived from the surgical medium by centrifugation. Samples were homogenized in a buffered medium containing protease inhibitor. Soluble and insoluble protein fractions were separated by centrifugation. The electrophoretic studies were performed according to Laemmli on equal amounts of proteins and were followed by silver intensification. Oxidized amino acid and Phe content of the samples were also analyzed by HPLC following acid hydrolysis of proteins.

Our results showed that soluble proteins represented a significantly lower portion of the total protein content in cataractous lenses in comparison with the control group (CONTR, 71.25%; non-DM CAT, 32.00%; DM CAT, 33.15%; p < 0.05 vs CONTR for both). Among the proteins, the crystallin-like proteins with low-molecular weight can be found both in the soluble and insoluble fractions, and high-molecular weight aggregates were found mainly in the total homogenates. In our HPLC analysis, oxidatively modified derivatives of phenylalanine were detected in cataractous samples. We found higher levels of m-Tyr, o-Tyr and DOPA in the total homogenates of cataractous samples compared to the supernatants. In all three groups, the median Phe/protein ratio of the total homogenates was also higher than that of the supernatants (total homogenates vs supernatants, in the CONTR group 1102 vs 633 μmol/g, in the DM CAT group 1187 vs 382 μmol/g and in the non-DM CAT group 967 vs 252 μmol/g; p < 0.05 for all).

In our study we found that oxidized amino acids accumulate in cataractous lenses, regardless of the origin of the cataract. The accumulation of the oxidized amino acids probably results from oxidation of Phe residues of the non-water soluble lens proteins. We found the presence of high-molecular weight protein aggregates in cataractous total homogenates, and a decrease of protein concentration in the water-soluble phase of cataractous lenses. The oxidation of lens proteins and the oxidative modification of Phe residues in key positions may lead to an altered interaction between protein and water molecules and thus contribute to lens opacification.     

Protein-bound kynurenine is a photosensitizer of oxidative damage

Human lens proteins become progressively modified by tryptophan-derived UV filter compounds in an age-dependent manner. One of these compounds, kynurenine, undergoes deamination at physiological pH, and the product binds covalently to nucleophilic residues in proteins via a Michael addition. Here we demonstrate that after covalent attachment of kynurenine, lens proteins become susceptible to photo-oxidation by wavelengths of light that penetrate the cornea. H2O2 and protein-bound peroxides were found to accumulate in a time-dependent manner after exposure to UV light (lambda > 305-385 nm), with shorter-wavelength light giving more peroxides. Peroxide formation was accompanied by increases in the levels of the protein-bound tyrosine oxidation products dityrosine and 3,4-dihydroxyphenylalanine, species known to be elevated in human cataract lens proteins. Experiments using D2O, which enhances the lifetime of singlet oxygen, and azide, a potent scavenger of this species, are consistent with oxidation being mediated by singlet oxygen. These findings provide a mechanistic explanation for UV light-mediated protein oxidation in cataract lenses, and also rationalize the occurrence of age-related cataract in the nuclear region of the lens, as modification of lens proteins by UV filters occurs primarily in this region.     

Temperature studies of glyceraldehyde-3-phosphate dehydrogenase binding to liposomes using fluorescence technique.

Interaction of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase with negatively charged liposomes was investigated as a function of temperature. This interaction affects the temperature-dependent conformational transition in the enzyme and exerts stabilizing effect on the protein structure. It can be seen from the fluorescence quenching experiments that the accessibility of tryptophanyl residues and isoindol probe fluorophores (covalently bound with the protein amino groups) for a dynamic quencher, acrylamide, is altered upon binding. This accessibility represented by effective quenching constant (Keff) strongly depends on temperature for unmodified enzyme and for the enzyme adsorbed on liposomes, it is nearly constant over a wide range of temperatures.     

Spectral studies on two genetic forms of the human serum proteinase inhibitor, alpha-1-antitrypsin.

alpha-1-antitrypsin, the major inhibitor of proteolytic enzymes in human serum, was isolated from normal individuals (protease inhibitor type MM) and from those with an inherited deficiency (protease inhibitor type ZZ) of circulatory protein. The two proteins were compared by circular dichroism spectroscopy, and by fluorescence quenching experiments using anionic (I-), and neutral (acrylamide) probes. Both proteins share a similar secondary structure, i.e. approximately 45--50% alpha-helix and 15--20% beta-structure. Evidence was accumulated to show that the microenvironment in the vicinity of the three tryptophanyl residues is altered in Z form as compared to the M form as shown by (a) the absence of the positive dichroic band in the region 290--300 nm of the circular dichroism spectra, (b) a greater than 50% increase in quantum yield in the tryptophanyl fluorescence emission spectra, (c) an increased accessibility of tryptophan to quenching by iodide, and (d) acrylamide quenching experiments which indicate that all tryptophanyl residues in the Z protein are quenched equally or that quenching is dominated by a single residue, while in the M protein, heterogeneous quenching occurs. The potential significance of these findings in terms of alpha-1-antitrypsin deficiency state are discussed.     

Structure elucidation of a novel yellow chromophore from human lens protein

We report here the isolation of a novel acid-labile yellow chromophore from the enzymatic digest of human lens proteins and the identification of its chemical structure by liquid chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, and (1)H, (13)C, and two-dimensional NMR. This new chromophore exhibited a UV absorbance maximum at 343 nm and fluorescence at 410 nm when excited at 343 nm. Analysis of the purified compound by reversed-phase HPLC with in-line electrospray ionization mass spectrometry revealed a molecular mass of 370 Da. One- and two-dimensional NMR analyses elucidated the structure to be 1-(5-amino-5-carboxypentyl)-4-(5-amino-5-carboxypentylamino)-3-hydroxy-2,3-dihydropyridinium, a cross-link between the epsilon-amino groups of two lysine residues, and a five-carbon ring. Because this cross-link contains two lysine residues and a dihydropyridinium ring, we assigned it the trivial name of K2P. Quantitative determinations of K2P in individual normal human lens or cataract lens water-soluble and water-insoluble protein digests were made using a high-performance liquid chromatograph equipped with a diode array detector. These measurements revealed a significant enhancement of K2P in cataract lens proteins (613 +/- 362 pmol/mg of water-insoluble sonicate supernatant (WISS) protein or 85 +/- 51 pmol/mg of WS protein) when compared with aged normal human lens proteins (261 +/- 93 pmol/mg of WISS protein or 23 +/- 15 pmol/mg of water-soluble (WS) protein). These data provide chemical evidence for increased protein cross-linking during aging and cataract development in vivo. This new cross-link may serve as a quantitatively more significant biomarker for assessing the role of lens protein modifications during aging and in the pathogenesis of cataract.     

Oxidative modification of lens crystallins by H2O2 and chelated iron

Crystallins are the soluble structural proteins that constitute approximately 90% of the dry mass of the eye lens. The present study attempts to elucidate possible mechanisms whereby the H2O2 present in the eye could contribute to the oxidative modification of lens crystallins. The data indicate that exposure of solutions of crystallins to H2O2 and EDTA-chelated iron leads to covalent crosslinking of polypeptides, loss of intrinsic protein fluorescence, and the generation of a novel fluorophor emitting in the 420 nm range. These changes closely mimic oxidative modifications that occur in lens proteins in vivo. Exposure of the proteins to H2O2 in the absence of chelated iron failed to generate detectable levels of these modifications. These findings are contrasted with earlier studies of lenses in organ culture where H2O2 alone produced marked damage while the further addition of chelated iron protected the lenses from oxidation.     

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.     

Development of fluorescence and cross-links in eye lens crystallin by interaction with lipid peroxy radicals

Senile nuclear cataract formation is associated with the accumulation of fluorescent insoluble proteins. alpha-Crystallin from bovine eye lens was treated with the lipid peroxy radicals generated by interaction of linoleic acid 13-monohydroperoxide or phosphatidylcholine hydroperoxide with methemoglobin. A blue non-tryptophan fluorescence composed of at least two kinds of fluorophores, stable and unstable on borohydride treatment, was produced. One of the stable fluorophores was identified as dityrosine by comparison of its retention times in amino acid analysis and HPLC with those of authentic dityrosine. Dityrosine was formed by the radical reaction, and less than 0.3% of the total tyrosine residues of the protein were transformed into dityrosine. The unstable fluorophores may be produced on the amino groups of alpha-crystallin by the non-radical reaction of the decomposition products of the radicals including monofunctional aldehyde species. Extensive intermolecular cross-links could not be explained only by the dityrosine content, but by the other modifications. Formation of fluorescence and intermolecular cross-links in alpha-crystalline in reaction with the lipid peroxy radicals suggested the participation of lipid peroxidation in the cataractous process.     

Mechanism of insolubilization by a single-point mutation in alphaA-crystallin linked with hereditary human cataracts

AlphaA-crystallin is a small heat shock protein that functions as a molecular chaperone and a lens structural protein. The R49C single-point mutation in alphaA-crystallin causes hereditary human cataracts. We have previously investigated the in vivo properties of this mutant in a gene knock-in mouse model. Remarkably, homozygous mice carrying the alphaA-R49C mutant exhibit nearly complete lens opacity concurrent with small lenses and small eyes. Here we have investigated the 90 degrees light scattering, viscosity, refractive index, and bis-ANS fluorescence of lens proteins isolated from the alphaA-R49C mouse lenses and found that the concentration of total water-soluble proteins showed a pronounced decrease in alphaA-R49C homozygous lenses. Light scattering measurements on proteins separated by gel permeation chromatography showed a small amount of high-molecular mass aggregated material in the void volume which still remains soluble in alphaA-R49C homozygous lens homogenates. An increased level of binding of beta- and gamma-crystallin to the alpha-crystallin fraction was observed in alphaA-R49C heterozygous and homozygous lenses but not in wild-type lenses. Quantitative analysis with the hydrophobic fluorescence probe bis-ANS showed a pronounced increase in fluorescence yield upon binding to alpha-crystallin from mutant as compared with the wild-type lenses. These results suggest that the decrease in the solubility of the alphaA-R49C mutant protein was due to an increase in its hydrophobicity and supra-aggregation of alphaA-crystallin that leads to cataract formation. Our study further shows that analysis of mutant proteins from the mouse model is an effective way to understand the mechanism of protein insolubilization in hereditary cataracts.     

Ion and water distribution in pig lenses incubated at 0 degree C to disable ion transport pumps.

This study was designed to test how extended exposure of lenses to sera with different ionic strengths influences the distribution of ions and water in the lens. Pig lenses were incubated in cold sera (0 degree C), which were adjusted to variable concentrations of NaCl, and their K+, Na+, Cl-, and water contents were measured. Incubation at 0 degree C inhibits active transport processes and thereby allows equilibration of the mobile ions and water. The hypothesis was that lens water content (volume) would follow the ion-induced protein changes predicted by a model derived from previous osmotic studies on proteins. As expected, exposure of the lens to cold caused a gain of sodium and a partial loss of potassium. However, the potassium concentration in the lens remained several fold higher than that in the bathing solution (about 41 vs. 1.8-4.6 mM/kg H2O), indicating that a portion of the potassium within the cold-exposed lens was not free to diffuse. That the water content of the lens showed a negative rather than a positive relationship with the concentration of NaCl within the lens was explained by the idea that an increase in NaCl within the lens (up to at least 250 mM/kg H2O) causes a decrease in the osmotically unresponsive water volume associated with lens proteins.     

Intracellular protein degradation in cultured bovine lens epithelial cells

Summary Although several proteases have been identified in homogenates of cultured epithelial cells of the eye lens and in lens tissues, there is little information regarding intracellular protein degradation in intact lens cells in vitro. Cultured lens cells may be useful in the study of intracellular protein degradation in the lens, a tissue with a wide range of protein half-lives. This is of interest because alterations in protein turnover in the lens have been implicated in cataract formation. This study examines intracellular protein degradation in cultured bovine lens epithelial cells (BLEC). Cell cultures were incubated with radiolabeled leucine to label intracellular proteins. Protein degradation was measured by monitoring the release of trichloroacetic-acid-soluble radioactivity into the culture medium. The average half-life of long-lived proteins (half-life >50 h) was typically about 57 h in serum-supplemented medium. Average rates of degradation of long-lived proteins increased by up to 73% when fetal bovine serum was withdrawn from the culture medium. Serum had no effect on the degradation of short-lived proteins (half-life <10 h). Degradation of long-lived proteins in the presence and absence of serum was further studied in cultured BLEC from population doubling level (PDL) 2 to 43. Average half-life of proteins in serum-supplemented medium was 52 to 58 h and did not vary significantly as a function of PDL. Degradation rates in serum-free medium increased approximately twofold up to PDL 7, but returned by PDL 25 to original levels, which were maintained through PDL 43. This work was supported in part by grants from U. S. Department of Agriculture contract 53-3K06-5-10, Massachusetts Lions Eye Research Fund, Inc., and the Daniel and Florence Guggenheim Foundation. D. A. E. is a recipient of a National Eye Institute postdoctoral fellowship.     

Determination of the accessibility and localization of tryptophan residues in the influenza virus hemagglutinin molecule

The accessibility and localization of tryptophane residues in the influenza viral hemagglutinin molecule have been determined by measuring specific quenching of tryptophane fluorescence by neutral (acrylamide), anionic (I-) and cationic (Cs+) quenchers. It has been shown that acrylamide quenches 64% of tryptophane fluorescence in H3-hemagglutinin whereas I- and Cs+ quench only 34%. The tryptophanyl residues have been assumed to be located in the hemagglutinin molecule both in the cationic and anionic environments. 64% of tryptophanyls have been shown to be located on the surface of the protein globule.     

Water permeability through biological membranes by isotopic effects of fluorescence and light scattering.

A light-scattering technique used to measure the water permeability across closed biomembranes is described, which is based on the different indices of refraction of D2O and H2O. This transient technique is compared with a similar method using D2O-sensitive fluorophores in the intravesicular space. The results of both techniques are equivalent although the signal-to-noise ratio favors the light-scattering or turbidity experiment. The light-scattering method is only applicable to larger particles (no point-scatterers) and is easily extended to biological objects. Data on the H2O/D2O exchange across membranes of ghosts from human erythrocytes suggest two mechanisms: the D2O and H2O permeation through the membrane and a slower D2O-induced conformational change of membraneous proteins.     

亚硒酸钠诱导的晶状体蛋白质聚合作用

不仅在体内,而且在体外亚硒酸钠可引起晶状体蛋白质聚合。将亚硒酸钠加到pH7.4的晶状体蛋白质溶液中,在37℃保温30min后观察到蛋白质溶液变混浊,随时间的延长混浊程度加重并有沉淀形成。经SDS聚丙烯酰胺凝胶电泳发现,加硒保温后形成的不溶性蛋白质中有大量的高分子聚合物。当加入二硫苏糖醇后混浊的蛋白质溶液变清,其中的高分子聚合物也基本消失,我们还发现;在亚硒酸钠使晶状体蛋白质变混浊的同时,蛋白质巯基减少,而蛋白质结合的硒量增加,且二者之间有较固定的比例关系,即蛋白质上每增加一个硒原子,蛋白质巯基就减少4.26个。当用二硫苏糖醇还原后,68％的硒从蛋白质中释放出来。这些结果表明,亚硒酸钠可引起大鼠晶状体水溶性蛋白质聚合,其可能方式如下:4PSH+SeO_3~-→PSSP+PS-Se-SP+H_2O+2OH~-这可能是亚硒酸钠诱发白内障的主要原因。     

Tryptophan-derived ultraviolet filter compounds covalently bound to lens proteins are photosensitizers of oxidative damage

The human eye is chronically exposed to light of wavelengths >300 nm. In the young human lens, light of wavelength 300-400 nm is predominantly absorbed by the free Trp derivatives kynurenine (Kyn), 3-hydroxykynurenine (3OHKyn), and 3-hydroxykynurenine-O-beta-D-glucoside (3OHKynG). These ultraviolet (UV) filter compounds are poor photosensitizers. With age, the levels of the free UV filters in the lens decreases and those of protein-bound UV filters increases. The photochemical behavior of these protein-bound UV filters and their role in UV damage are poorly elucidated and are examined here. UVA illumination of protein-bound UV filters generated peroxides (principally H2O2) in a metabolite-, photolysis-time-, and wavelength-dependent manner. Unmodified proteins, free Trp metabolites, and Trp metabolites that do not bind to lens proteins gave low peroxide yields. Protein-bound 3OHKyn (principally at Cys residues) yielded more peroxide than comparable Kyn and 3OHKynG adducts. Studies using D2O and sodium azide implicated 1O2 as a key intermediate. Illumination of the protein-bound adducts also yielded protein-bound Tyr oxidation products (DOPA, di-tyrosine) and protein cross-links via alternative mechanisms. These data indicate that the covalent modification of lens proteins by Kyn derivatives yields photosensitizers that may enhance oxidation in older lenses and contribute to age-related nuclear cataract.     

Elucidation of intermediate (mobile) and slow (solidlike) protein motions in bovine lens homogenates by carbon-13 NMR spectroscopy

The motional dynamics of lens cytoplasmic proteins present in calf lens homogenates were investigated by two 13C nuclear magnetic resonance (NMR) techniques sensitive to molecular motion to further define the organizational differences between the cortex and nucleus. For the study of intermediate (mobile) protein rotational reorientation motion time scales [rotational correlation time (tau 0) range of 1-500 ns], we employed 13C off-resonance rotating frame spin-lattice relaxation, whereas for the study of slow (solidlike) motions (tau 0 greater than or equal to 10 microseconds) we used the solid-state NMR techniques of dipolar decoupling and cross-polarization. The frequency dependence of the peptide bond carbonyl off-resonance rotating frame spectral intensity ratio of the lens proteins present in native calf nuclear homogenate (42% protein) at 35 degrees C indicates the presence of a polydisperse mobile protein fraction with a tau 0,eff (mean) value of 57 ns. This mean value is consistent with the average value calculated from the known water-soluble nuclear lens protein polydispersity assuming a cytoplasmic viscosity 3 times that of pure water. Lowering the temperature to 1 degree C, a temperature which produces the cold cataract, results in an overall decrease in tau 0,eff to 43 ns, suggesting a selective removal of beta H-, LM-, and possibly gamma s-crystallins from the mobile lens protein population. The presence of solidlike or motionally restricted protein species was established by dipolar decoupling and cross-polarization. The fraction of motionally restricted protein in the nuclear region varied from 0.35 to 0.45 in the temperature range of 35-1 degree C. For native cortical homogenate (25% protein), the off-resonances rotating frame spectral intensity ratio frequency-dependent curves for the protein carbonyl resonance yielded tau 0,eff values of 34 and 80 ns at 35 and 1 degree C, respectively. Both values were reconciled with the known lens cortex soluble protein polydispersity using an assumed cytoplasmic viscosity 1.5 times that of pure water at the same temperature. Comparison of proton dipolar-decoupled and nondecoupled 13C NMR spectra of native cortical homogenate at 20 degrees C indicates the absence of significant contributions from slowly tumbling, motionally restricted species. This interpretation was confirmed by the failure to detect significant lens protein 13C-1H cross-polarization at this temperature. However, at 1 degree C, the fraction of solidlike protein was 0.15. Concentrated cortical homogenates at 20 degrees C (42% protein), by contrast, gave cross-polarization spectra with maximum absolute signal intensities 50-70% of native nuclear homogenates, but with similar magnetization parameters...     

The rapid H2O2-mediated nonphotodynamic crosslinking of lens crystallins generated by the heme-undecapeptide from cytochrome C: potential implications for cataractogenesis in man

Human cataract lens crystallins are crosslinked and demonstrate a non-tryptophan blue fluorescence. We report here that exposure of lens crystallin to H2O2 within the concentration range reported for human aqueous humor, produces crosslinking of crystallin polypeptides within 10 minutes in the presence of the heme-undecapeptide from cytochrome c. Concomitantly, a blue fluorescence develops. These findings suggest the possibility that under some conditions hydrogen peroxide activation may play a role in cataractogenesis in vivo.     

Rate of formation of AGEs during ascorbate glycation and during aging in human lens tissue

The similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid modified calf lens proteins was recently published [Biochim. Biophys. Acta 1537 (2001) 14]. The data presented here additionally quantify age-dependent increases in individual yellow chromophores and fluorophores in the water-insoluble fraction of normal human lens. The water-insoluble fraction of individual normal human lens was isolated, solubilized by sonication and digested with a battery of proteolytic enzymes under argon to prevent oxidation. The level of A(330)-absorbing yellow chromophores, 350/450 nm fluorophores and total water-insoluble (WI) protein were quantified in each lens. The total yellow chromophores and fluorophores accumulated in parallel with the increase in the water-insoluble protein fraction during aging. The digest from each single human lens was then subjected to Bio-Gel P-2 size-exclusion chromatography. The fractions obtained were further separated by a semi-preparative prodigy C-18 high-performance liquid chromatography (RP-HPLC). Bio-Gel P-2 chromatography showed four major fractions, each of which increased with age. RP-HPLC of the amino acid peak resolved five major A(330)-absorbing peaks and eight fluorescent peaks, and each peak increased coordinately with age. A late-eluting peak, which contained hydrophobic amino acids increased significantly after age 60.Aliquots from an in vitro glycation of calf lens proteins by ascorbic acid were removed and subjected to the same enzymatic digestion. Ascorbic acid-modified calf lens protein digests showed an almost identical profile of chromophores, which also increased in a time-dependent manner. The late-eluting peak, however, did not increase with the time of glycation and may not be an advanced glycation endproduct (AGE) product. The data indicate that the total water-insoluble proteins, individual yellow chromophores and fluorophores increased equally both with aging in normal human lens and during ascorbate glycation in vitro. The major protein modifications, which accumulate during aging, therefore, appear to be AGEs. Whereas the late-eluting peak, which showed poor correlation to ascorbylation, may represent UV filter compounds bound to lens proteins.