Human Eye Lens Research Articles

Influence of Contact Lens Parameters on Cornea: Biomechanical Analysis.

This study presents a finite element analysis to model ocular biomechanics and the interactions between the human eye and contact lenses in the closed-eye condition. The closed-eye state, where the eyelids are fully shut, presents challenges for experimental measurements due to the invasive nature of accessing and analysing the contact lens and corneal interface, making simulation tools valuable for accurate characterisation. The primary objective of this study was to examine how CLs fold and twist and their impact on the cornea when the eye is closed. The secondary aim of this study was to assess how crucial contact lens parameters (Young's modulus, base curve, and diameter) influence corneal stress distribution and the overall fit of the lens on the eye. The findings show that increasing Young's modulus significantly reduces corneal stress and promotes uniform stress distribution, making it the most influential factor for wearer comfort and safety. While base curve and diameter variations primarily affect contact area, their impact on stress distribution is minimal. This research provides insights for improving contact lens design and enhancing safety for contact lens wearers.

Structure of the lens MP20 mediated adhesive junction.

Human lens fiber membrane intrinsic protein MP20 is the second most abundant membrane protein of the human eye lens. Despite decades of effort its structure and function remained elusive. Here, we determined the MicroED structure of full-length human MP20 in lipidic-cubic phase to a resolution of 3.5 Å. MP20 forms tetramers each of which contain 4 transmembrane α-helices that are packed against one another forming a helical bundle. Both the N- and C- termini of MP20 are cytoplasmic. We found that each MP20 tetramer formed adhesive interactions with an opposing tetramer in a head-to-head fashion. These interactions were mediated by the extracellular loops of the protein. The dimensions of the MP20 adhesive junctions are consistent with the 11 nm thin lens junctions. Investigation of MP20 localization in human lenses indicated that in young fiber cells MP20 was stored intracellularly in vesicles and upon fiber cell maturation MP20 inserted into the plasma membrane and restricted the extracellular space. Together these results suggest that MP20 forms lens thin junctions in vivo confirming its role as a structural protein in the human eye lens, essential for its optical transparency.

Entropy Tug-of-War Determines Solvent Effects in the Liquid-Liquid Phase Separation of a Globular Protein.

Liquid-liquid phase separation (LLPS) plays a key role in the compartmentalization of cells via the formation of biomolecular condensates. Here, we combined atomistic molecular dynamics (MD) simulations and terahertz (THz) spectroscopy to determine the solvent entropy contribution to the formation of condensates of the human eye lens protein γD-Crystallin. The MD simulations reveal an entropy tug-of-war between water molecules that are released from the protein droplets and those that are retained within the condensates, two categories of water molecules that were also assigned spectroscopically. A recently developed THz-calorimetry method enables quantitative comparison of the experimental and computational entropy changes of the released water molecules. The strong correlation mutually validates the two approaches and opens the way to a detailed atomic-level understanding of the different driving forces underlying the LLPS.

Cholesterol Content Regulates the Interaction of αA-, αB-, and α-Crystallin with the Model of Human Lens-Lipid Membranes.

α-Crystallin (αABc) is a major protein comprised of αA-crystallin (αAc) and αB-crystallin (αBc) that is found in the human eye lens and works as a molecular chaperone by preventing the aggregation of proteins and providing tolerance to stress. However, with age and cataract formation, the concentration of αABc in the eye lens cytoplasm decreases, with a corresponding increase in the membrane-bound αABc. This study uses the electron paramagnetic resonance (EPR) spin-labeling method to investigate the role of cholesterol (Chol) and Chol bilayer domains (CBDs) in the binding of αAc, αBc, and αABc to the Chol/model of human lens-lipid (Chol/MHLL) membranes. The maximum percentage of membrane surface occupied (MMSO) by αAc, αBc, and αABc to Chol/MHLL membranes at a mixing ratio of 0 followed the trends: MMSO (αAc) > MMSO (αBc) ≈ MMSO (αABc), indicating that a higher amount of αAc binds to these membranes compared to αBc and αABc. However, with an increase in the Chol concentration in the Chol/MHLL membranes, the MMSO by αAc, αBc, and αABc decreases until it is completely diminished at a mixing ratio of 1.5. The Ka of αAc, αBc, and αABc to Chol/MHLL membranes at a mixing ratio of 0 followed the trend: Ka (αBc) ≈ Ka (αABc) > Ka (αAc), but it was close to zero with the diminished binding at a Chol/MHLL mixing ratio of 1.5. The mobility near the membrane headgroup regions decreased with αAc, αBc, and αABc binding, and the Chol antagonized the capacity of the αAc, αBc, and αABc to decrease mobility near the headgroup regions. No significant change in membrane order near the headgroup regions was observed, with an increase in αAc, αBc, and αABc concentrations. Our results show that αAc, αBc, and αABc bind differently with Chol/MHLL membranes at mixing ratios of 0 and 0.5, decreasing the mobility and increasing hydrophobicity near the membrane headgroup region, likely forming the hydrophobic barrier for the passage of polar and ionic molecules, including antioxidants (glutathione), creating an oxidative environment inside the lens, leading to the development of cataracts. However, all binding was completely diminished at a mixing ratio of 1.5, indicating that high Chol and CBDs inhibit the binding of αAc, αBc, and αABc to membranes, preventing the formation of hydrophobic barriers and likely protecting against cataract formation.

Revamped mini-αA-crystallin showed improved skin permeation and therapeutic activity against melittin-induced toxicity

Melittin is honey bee venom's primary and most toxic pharmacologically active component. Melittin causes haemolysis, lymphocyte lysis, long-term pain, localised inflammation followed by rhabdomyolysis, and severe renal failure. Renal failure or cardiovascular complications could lead to the victim's death. Severe honey bee bites are treated with general medication involving antihistaminic, anti-inflammatory, and analgesic drugs, as a specific treatment option is unavailable. An earlier study showed the anti-hemolysis and anti-lymphocyte lysis activity of mini- αA-crystallin (MAC), a peptide derived from human eye lens alpha-crystallin. MAC's use has often been restricted despite its high therapeutic potential due to its poor skin permeability. This study compared the skin permeation, anti-inflammatory and analgesic activities of natural peptide MAC and its modified version (MAC-GRD) formed by attaching cell-penetrating peptide (CPP) and GRD amino residues into MAC. Gel formulations were prepared for MAC and MAC-GRD peptides using carbopol (1% w/w), Tween 80 (1%), and ethanol (10%). An ex-vivo skin permeation study was performed using a vertical-type Franz diffusion apparatus. Preclinical in-vivo experiments were conducted to compare the native and modified peptide formulations against melittin-induced toxicity in Wistar rats. MAC gel, MAC–GRD gel and 1% hydrocortisone cream significantly reduced the melittin-induced writhing (20.16 ± 0.792) response in rats with 15.16 ± 0.47, 11.16 ± 0.477 and 12.66 ± 0.66 wriths, respectively. There was a significant reduction in melittin-induced inflammation when MAC-GRD gel was applied immediately after melittin administration. At 0.5, 1, 3, and 5 h, the MAC-GRD-treated rat paws were 0.9 ± 0.043 mm, 0.750 ± 0.037 mm, 0.167 ± 0.0070 mm, and 0.133 ± 0.031 mm thick. Administration of melittin resulted in reduced GSH (antioxidant) levels (47.33 ± 0.760 μg/mg). However, treatment with MAC-GRD gel (71.167 ± 0.601 μg/mg), MAC gel (65.167 ± 1.138 μg/mg), and 1% hydrocortisone (68.33 ± 0.667 μg/mg) significantly increased the antioxidant enzyme levels. MAC-GRD gel significantly reduced the elevated MDA levels (6.933 ± 0.049 nmol/mg) compared to the melittin group (12.533 ± 0.126 nmol/mg), followed by the 1% hydrocortisone (7.367 ± 0.049 nmol/mg) and MAC gel (7.917 ± 0.048 nmol/mg). MAC-GRD demonstrated more skin permeability and superior anti-inflammatory, analgesic, and antioxidant activities when compared to MAC gel. When compared to standard 1% hydrocortisone cream, MAC-GRD had better anti-inflammatory, analgesic, antioxidant, and comparable action in anti-oxidant restoration against melittin. These findings suggest that the developed MAC-GRD gel formulation could help to treat severe cases of honey bee stings.

Противодействие живых систем развитию возрастной катаракты. Физико-химическое объяснение

Simultaneous processes occur in living systems that lead to both an increase in entropy (chaos) and its decrease. A decrease in entropy is possible only in open systems, associated with a contin-uous flow of energy-intensive compounds into them, such as glucose, the oxidation of which is energetically associated with the synthesis of ATP, the universal carrier of chemical energy to en-sure functioning of living systems. When energy-intensive compounds are oxidized, entropy in-creases, and when ATP is synthesized, it decreases. In living systems, not only reactions associated with the synthesis of ATP and their functioning occur, but also aging does, namely, the oxidation of hydrophobic formations in cells, in particular, the membrane matrix, following the type of chain reactions with degenerate branching. The development of such chain reactions of oxidation in cells can be prevented by the penetration into them of various antioxidants, for example, resvera-trol, which is contained in grapes and other plants, and quenchers of molecular oxygen in the sin-glet state, for example, iodides, as well as by creating cyclic processes involving desaturases that prevent an increase in viscosity in the matrix of membranes up to certain limiting values, after which cell death becomes inevitable. In this regard, the processes leading to cataracts (clouding of the eye lenses) are indicative. In the absence of desaturases in the lens fibers of rats, exposure to UV light leads to cataracts in 2–4 months, while the presence of desaturases in human eye lens fi-bers makes it possible to avoid the occurrence of cataracts for many decades. The reaction of con-version of lipids with saturated fatty acids into unsaturated ones, catalyzed by desaturases, leads to counteracting the aging of the membrane matrix. The vital feature of this reaction is that it oc-curs only when the membrane matrix is cured. This reaction first leads to liquefaction of the ma-trix up to a certain state, after which it stops. The reasons leading either to revival of desaturase or to decrease in its activity are discussed in this article.

Copper Reductase Activity and Free Radical Chemistry by Cataract-Associated Human Lens γ-Crystallins.

Cataracts are caused by high-molecular-weight aggregates of human eye lens proteins that scatter light, causing lens opacity. Metal ions have emerged as important potential players in the etiology of cataract disease, as human lens γ-crystallins are susceptible to metal-induced aggregation. Here, the interaction of Cu2+ ions with γD-, γC-, and γS-crystallins, the three most abundant γ-crystallins in the lens, has been evaluated. Cu2+ ions induced non-amyloid aggregation in all three proteins. Solution turbidimetry, sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS-PAGE), circular dichroism, and differential scanning calorimetry showed that the mechanism for Cu-induced aggregation involves: (i) loss of β-sheet structure in the N-terminal domain; (ii) decreased thermal and kinetic stability; (iii) formation of metal-bridged species; and (iv) formation of disulfide-bridged dimers. Isothermal titration calorimetry (ITC) revealed distinct Cu2+ binding affinities in the γ-crystallins. Electron paramagnetic resonance (EPR) revealed two distinct Cu2+ binding sites in each protein. Spin quantitation demonstrated the reduction of γ-crystallin-bound Cu2+ ions to Cu+ under aerobic conditions, while X-ray absorption spectroscopy (XAS) confirmed the presence of linear or trigonal Cu+ binding sites in γ-crystallins. Our EPR and XAS studies revealed that γ-crystallins' Cu2+ reductase activity yields a protein-based free radical that is likely a Tyr-based species in human γD-crystallin. This unique free radical chemistry carried out by distinct redox-active Cu sites in human lens γ-crystallins likely contributes to the mechanism of copper-induced aggregation. In the context of an aging human lens, γ-crystallins could act not only as structural proteins but also as key players for metal and redox homeostasis.

Early Stage UV-B Induced Molecular Modifications of Human Eye Lens γD-Crystallin.

In the human eye lenses, the crystallin proteins facilitate transparency, light refraction, as well as UV light protection. A deregulated balanced interplay between α-, β-, and γ-crystallin can cause cataract. γD-crystallin (hγD) is involved in the energy dissipation of absorbed UV light by energy transfer between aromatic side chains. Early UV-B induced damage of hγD with molecular resolution is studied by solution NMR and fluorescence spectroscopy. hγD modifications are restricted to Tyr 17 and Tyr 29 in the N-terminal domain, where a local unfolding of the hydrophobic core is observed. None of the tryptophan residues assisting fluorescence energy transfer is modified and hγD is remained soluble over month. Investigating isotope-labeled hγD surrounded by eye lens extracts from cataract patients reveals very week interactions of solvent-exposed side chains in the C-terminal hγD domain and some remaining photoprotective properties of the extracts. Hereditary E107A hγD found in the eye lens core of infants developing cataract shows under the here used conditions a thermodynamic stability comparable to the wild type but an increased sensitivity toward UV-B irradiation.

Conformational stability of the deamidated and mutated human βB2-crystallin

Previous studies propose that genetic mutations and post-translational modifications in protein crystallins promote protein aggregation and are considered significant risk factors for cataract formation. The βB2-crystallin (HβB2C) forms a high proportion of proteins in the human eye lens. Different congenital mutations and post-translational deamidations in βB2-crystallin have been reported and linked to cataract formation. In this work, we employed extensive all-atom molecular dynamics simulations to evaluate the conformational stability of deamidated and mutated HβB2C. Our results show critical changes in the protein surface and its native contacts due to a modification in the conformational equilibrium of these proteins. The double deamidated (Q70E/Q162E) and single deamidated (Q70E) impact the well compact conformation of the HβB2C. These post-translational modifications allow the exposure of the protein hydrophobic interface, which lead to the exposure of electronegative residues. On the other hand, our mutational studies showed that the S143F mutation modifies the hydrogen-bond network of an antiparallel β-sheet, unfolding the C-terminal domain. Interestingly, the chain termination mutation (Q155X) does not unfold the N-terminal domain. However, the resultant conformation is more compact and avoids the exposure of the hydrophobic interface. Our results provide valuable information about the first steps of HβB2C unfolding in the presence of deamidated amino acids that have been reported to appear during aging. The findings reported in this work are essential for the general knowledge of the initial steps in the cataract formation mechanism, which may be helpful for the further development of molecules with pharmacological potential against cataract disease.

A native chemical chaperone in the human eye lens suppresses redox-dependent lens crystallin misfolding.

Light-scattering protein aggregates in the eye lens cause cataract, one of the most prevalent protein aggregation disorders and still the most common cause of vision loss worldwide. The metabolically quiescent core region of the human lens lacks cellular or protein turnover and has therefore evolved remarkable mechanisms to resist protein aggregation for a lifetime. Lens cytoplasm becomes progressively more oxidizing with age and especially with cataract onset. Proteomic and in vitro studies have converged on the importance of lens γ-crystallin misfolding via formation of non-native disulfide bonds for light scattering aggregation. We have now found that one of the evolved mechanisms of lens resilience involves an unusually abundant lens metabolite, myo-inositol, that suppresses oxidative γ-crystallin aggregation without being redox-active itself. We quantified aggregation suppression using our previously well-characterized in vitro aggregation assays of oxidation-mimicking human γD-crystallin variants and investigated myo­inositol's molecular mechanism of action using solution NMR, negative-stain TEM, differential scanning fluorometry, thermal scanning Raman spectroscopy, turbidimetry in redox buffers, and free thiol quantitation. Unlike many known chemical chaperones, myo-inositol's primary target was not the native, unfolded, or final aggregated states of the protein; rather, we propose that it was the rate-limiting bimolecular step on the aggregation pathway. Given recent metabolomic evidence that myo-inositol is severely depleted in human cataractous lenses compared to age-matched controls, maintaining or restoring healthy levels of this compound in the lens may be a simple, safe, and globally accessible strategy to prevent or delay lens opacification due to age-onset cataract.

The Visual Quality of Healthy Eye Lens for Different Tilt Effects

The importance and accuracy of the human visual system have led scientists and researchers to do their best to study it, know its problems, and how to treat them. The eye lens tilt is possibly due to a specific accident such as a blow or the like. So, this manuscript was submitted to study the human eye lens tilting effect on visual performance and determine the tolerance tilt values. Using (ZEMAX-EE) software program and relying on the Liou and Brennan model Eye (LBME), one of the most accurate models for studying the eye, and some basic criteria used to describe the image formed in an optical system, such as Modulation Transfer Function (MTF), Root Main Square-spot size (RMSS), and simulated image including aberration, the study of the lens tilt effect on visual quality was conducted. The calculation of the area under the MTF curve was also used to determine the tolerance tilt values. Where the lens was tilted from (-5 to 5) degrees by (1 degree) on the (X and Y) axes, for zero decentering, with visible wavelength (0.486, 0.587, 0.656) µm, pupil diameter (2.5 mm), and field of view (5o).The results were that vision worsened when the lens was tilted due to reduced MTF. Sagittal MTF was less sensitive than Tangential due to the nature of the lens structure. The area under the MTF curve results also proved that the tilt is less than (5) degrees does not affect the visual much, so it was necessary to determine the tilt tolerance values.It was concluded that the visual quality was inversely proportional to the tilt increase. Exceeding the tilt (5) degrees led to visual deterioration due to increased optical aberration where the tolerance tilt ranges were (± 5.1) degrees and (- 5.9 to 9) degrees on the (x and y) axes, respectively.

A native chemical chaperone in the human eye lens.

Cataract is one of the most prevalent protein aggregation disorders and still the most common cause of vision loss worldwide. The metabolically quiescent core region of the human lens lacks cellular or protein turnover; it has therefore evolved remarkable mechanisms to resist light-scattering protein aggregation for a lifetime. We now report that one such mechanism involves an unusually abundant lens metabolite, myo-inositol, suppressing aggregation of lens crystallins. We quantified aggregation suppression using our previously well-characterized in vitro aggregation assays of oxidation-mimicking human γD-crystallin variants and investigated myo-inositol's molecular mechanism of action using solution NMR, negative-stain TEM, differential scanning fluorometry, thermal scanning Raman spectroscopy, turbidimetry in redox buffers, and free thiol quantitation. Unlike many known chemical chaperones, myo-inositol's primary target was not the native, unfolded, or final aggregated states of the protein; rather, we propose that it was the rate-limiting bimolecular step on the aggregation pathway. Given recent metabolomic evidence that it is severely depleted in human cataractous lenses compared to age-matched controls, we suggest that maintaining or restoring healthy levels of myo-inositol in the lens may be a simple, safe, and globally accessible strategy to prevent or delay lens opacification due to age-onset cataract.

Physics-aware learning and domain-specific loss design in ophthalmology.

The human cataract, a developing opacification of the human eye lens, currently constitutes the world's most frequent cause for blindness. As a result, cataract surgery has become the most frequently performed ophthalmic surgery in the world. By removing the human lens and replacing it with an artificial intraocular lens (IOL), the optical system of the eye is restored. In order to receive a good refractive result, the IOL specifications, especially the refractive power, have to be determined precisely prior to surgery. In the last years, there has been a body of work to perform this prediction by using biometric information extracted from OCT imaging data, recently also by machine learning (ML) methods. Approaches so far consider only biometric information or physical modelling, but provide no effective combination, while often also neglecting IOL geometry. Additionally, ML on small data sets without sufficient domain coverage can be challenging. To solve these issues, we propose OpticNet, a novel optical refraction network based on an unsupervised, domain-specific loss function that explicitly incorporates physical information into the network. By providing a precise and differentiable light propagation eye model, physical gradients following the eye optics are backpropagated into the network. We further propose a new transfer learning procedure, which allows the unsupervised pre-training on the optical model and fine-tuning of the network on small amounts of surgical patient data. We show that our method outperforms the current state of the art on five OCT-image based data sets, provides better domain coverage within its predictions, and achieves better physical consistency.

Accelerated oxidative aging in deamidated variants of the human eye lens protein γ(S)-crystallin

Cataract disease causes clouding of the eye lens due to precipitation of lens proteins, known as crystallins. In aged and cataractous lenses, crystallins have been shown to accumulate post-translational modifications (PTMs), with deamidation and oxidation among the most commonly reported PTMs. Both deamidation and oxidation appear to promote crystallin aggregation; however, the relationship between these two modifications and their role in crystallin insolubility is not clear. We determined six novel crystal structures of a major human lens protein, γS-crystallin (γS): one of the wild-type in a monomeric state, and five of deamidated γS variants, ranging from three to nine deamidation sites, after varying degrees of sample aging.

Disruption of a conserved N-terminal IXI motif of αB-crystallin leads to the formation of dynamic fibrillar aggregates

Proteostasis of the human eye lens is supported by a pair of small heat shock proteins (sHSPs), known as α-crystallins ( αA- and αB-isoforms). These proteins function as polydisperse multimers that sequester destabilized proteins and prevent them from aggregating in an ATP-independent manner (aka a “holdase”). In the eye lens, these features are thought to prevent the formation of light-scattering aggregates that give rise to age-related cataract. The functional polydispersity and chaperone function of the α-crystallins are mediated by intrinsically disordered regions located at the protein’s n-terminal domain (NTD) and c-terminal extension (CTE).

Human γS-Crystallin Resists Unfolding Despite Extensive Chemical Modification from Exposure to Ionizing Radiation.

Ionizing radiation has dramatic effects on living organisms, causing damage to proteins, DNA, and other cellular components. γ radiation produces reactive oxygen species (ROS) that damage biological macromolecules. Protein modification due to interactions with hydroxyl radical is one of the most common deleterious effects of radiation. The human eye lens is particularly vulnerable to the effects of ionizing radiation, as it is metabolically inactive and its proteins are not recycled after early development. Therefore, radiation damage accumulates and eventually can lead to cataract formation. Here we explore the impact of γ radiation on a long-lived structural protein. We exposed the human eye lens protein γS-crystallin (HγS) to high doses of γ radiation and investigated the chemical and structural effects. HγS accumulated many post-translational modifications (PTMs), appearing to gain significant oxidative damage. Biochemical assays suggested that cysteines were affected, with the concentration of free thiol reduced with increasing γ radiation exposure. SDS-PAGE analysis showed that irradiated samples form protein-protein cross-links, including nondisulfide covalent bonds. Tandem mass spectrometry on proteolytic digests of irradiated samples revealed that lysine, methionine, tryptophan, leucine, and cysteine were oxidized. Despite these chemical modifications, HγS remained folded past 10.8 kGy of γ irradiation as evidenced by circular dichroism and intrinsic tryptophan fluorescence spectroscopy.

Immunohistochemical Expression of Alpha (Α) A Crystallin in Senile Degenerative and Non-Cataract Lenses

Aim: Comparative immunohistochemical study of expression of α A Crystallin in non-cataract lenses and age-related cataract lenses in humans. Methodology: This was an observational cross sectional study. There are two groups in this study. Group A comprised of 121 senile degenerative cataract lenses from diagnosed patients. Group B included of 10 non-cataract lenses from patients who underwent surgeries for enucleation due to trauma and retinoblastoma. Lenses were fixed in 10% Buffered Neutral Formalin and processed to make paraffin blocks. Immunohistochemistry (IHC) staining was performed on sections using primary antibody for α A crystallin. Data was analyzed through SPSS software version 24. Results: Immunohistochemical staining of group A showed 80.2% Strong Positive expression while 19.8% showed Intermediate Positive expression of α A Crystallin. 100% Strong Positive expression of α A Crystallin was seen in group B. Comparison of expression of α A Crystallin in two groups showed significant decrease (p<0.001) in expression. Conclusion: Decreased expression of α A Crystallin in IHC stained senile cataract lens indicates the role of structural alterations of lens fibers in pathogenesis of senile cataract. If mechanism involved in causing these alterations can be identified and targeted so that progression of senile cataract may be delayed. Keywords: Immunohistochemistry, α A crystallin expression, senile cataract, Human eye Lens, Lens Fiber.

Influence of pH on radical reactions between kynurenic acid and amino acids tryptophan and tyrosine. Part II. Amino acids within the protein globule of lysozyme

An acidosis, a decrease of pH within a living tissue, may alter yields of radical reactions if participating radicals undergo partial or complete protonation. One of photosensitizers found in the human eye lens, kynurenic acid (KNA−), possesses pKa 5.5 for its radical form that is close to physiological pH 6.89 for a healthy lens. In this work we studied the influence of pH on mechanisms and products of photoinduced radical reactions between KNA− and amino acids tryptophan (Trp) and tyrosine (Tyr) within a globule of model protein, Hen White Egg Lysozyme (HEWL). Our results show that the rate constant of back electron transfer from kynurenyl to HEWL• radicals with the restoration of initial reagents – the major decay pathway for these radicals – does not change in the pH 3–7. The quantum yield of HEWL degradation is also pH independent, however a shift of pH from 7 to 5 completely changes the outcome of photoinduced damage to HEWL from intermolecular cross-linking to oxygenation. HPLC-MS analysis has shown that four of six Trp and all Tyr residues of HEWL are modified in different extents at all pH, but the lowering of pH from 7 to 5 significantly changes the direction of main photodamage from Trp62 to Trp108 located at the entrance and bottom of enzymatic center, respectively. A decrease of intermolecular cross-links via Trp62 is followed by an increase in quantities of intramolecular cross-links Tyr20-Tyr23 and Tyr23-Tyr53. The obtained results point out the competence of cross-linking and oxygenation reactions for Trp and Tyr radicals within a protein globule and significant increase of oxygenation to the total damage of protein in the case of cross-linking deceleration by coulombic repulsion of positively charged protein globules.

Influence of pH on radical reactions between kynurenic acid and amino acids tryptophan and tyrosine. Part I. Amino acids in free state

In the human eye lens the endogenous chromophores of UV-A light (315–400 nm) are able to sensitize radical reactions leading to protein modifications during normal aging and the cataract progression. Kynurenic acid (KNA−) is the most photochemically active dye of the human eye lens reported to date with pKa(KNAH2•) 5.5 for its radical form. Cataract is thought to develop under oxidative stress which could be accompanied by acidosis, an acidification of the intracellular environment. Protonation of kynurenyl radicals at mildly acidic conditions may change the outcome of radical reactions leading to additional damage to proteins. In this work we investigated the influence of pH on the degradation of initial reagents and the formation of products in photoinduced radical reactions between KNA− and amino acids tryptophan (Trp) and tyrosine (Tyr) in free states. Our results have shown that pH variation has minor influence on kinetics of reagent decay and accumulation of products in reactions between tyrosyl and kynurenic acid radicals. However in the case of Trp a two-fold decrease of the reagent degradation without visible changes in the composition of formed products was observed with pH decrease from 7 to 3. Time-resolved measurements have shown similar acidification-induced two-fold acceleration of decay of kynurenyl and tryptophanyl radicals via Back Electron Transfer (BET) with the restoration of initial reagents. Experiments with tryptophan derivatives with different pKa values for their radical forms point out the protonation of tryptophanyl radical as the driving force for BET acceleration at low pH. Our results demonstrate that the protonation of kynurenyl radical does not change its reactivity towards amino acids radicals but the total yield of radical photodamage decreases with the protonation of tryptophanyl radicals. It could be expected that radical induced damage to proteins will depend on the pKa of tryptophanyl radicals within a protein globule.

Protection of human γD-crystallin protein from ultraviolet C-induced aggregation by ortho-vanillin

Cataract is known as one of the leading causes of vision impairment worldwide. While the detailed mechanism of cataratogenesis remains unclear, cataract is believed to be correlated with the aggregation and/or misfolding of human ocular lens proteins called crystallins. A 173-residue structural protein human γD-crystallin is a major γ-crystallin protein in the human eye lens and associated with the development of juvenile and mature-onset cataracts. This work is aimed at investigating the effect of a small molecule, e.g., ortho-vanillin, on human γD-crystallin aggregation upon exposure to ultraviolet-C irradiation. According to the findings of right-angle light scattering, transmission electron microscopy, and gel electrophoresis, ortho-vanillin was demonstrated to dose-dependently suppress ultraviolet-C-triggered aggregation of human γD-crystallin. Results from the synchronous fluorescence spectroscopy, tryptophan fluorescence quenching, and molecular docking studies revealed the structural change of γD-crystallin induced by the interaction/binding between ortho-vanillin and protein. We believe the outcome from this work may contribute to the development of potential therapeutics for cataract.