Investigation of the peptides with calcium chelating capacity in hydrolysate derived from spent hen meat.

Calcium peptide chelates are developed as efficient supplements for preventing calcium deficiency. Spent hen meat (SHM) contains a high percentage of proteins but is generally wasted due to the disadvantages such as hard texture. We chose the underutilized SHM to produce peptides to bind calcium by proteolysis and aimed to investigate chelation between calcium and peptides in hydrolysate for a sustainable purpose. The optimized proteolysis conditions calculated from the result of response surface methodology for two-step hydrolysis were 0.30% (wenzyme/wmeat) for papain with a hydrolysis time of 3.5 h and 0.18% (wenzyme/wmeat) for flavourzyme with a hydrolysis time of 2.8 h. The enzymatic hydrolysate (EH) showed a binding capacity of 63.8 ± 1.8 mg calcium/g protein. Ethanol separation for EH improved the capacity up to a higher value of 68.6 ± 0.6 mg calcium/g protein with a high association constant of 420 M-1 (25°C) indicating high stability. The separated fraction with a higher amount of Glu, Asp, Lys, and Arg had higher calcium-binding capacity, which was related to the number of ─COOH and ─NH2 groups in peptide side chains according to the result from amino acid analysis and Fourier transform infrared spectroscopy. Two-step enzymatic hydrolysis and ethanol separation were an efficient combination to produce peptide mixtures derived from SHM with high calcium-binding capacity. The high percentage of hydrophilic amino acids in the separated fraction was concluded to increase calcium-binding capacity. This work provides foundations for increasing spent hen utilization and developing calcium peptide chelates based on underutilized meat.

Calcium bioaccessibility increased during gastrointestinal digestion of α-lactalbumin and ß-lactoglobulin.

Calcium bioaccessibility depends on the amount of soluble calcium under intestinal digestion. The changes in calcium during in vitro static digestion of α-lactalbumin and ß-lactoglobulin in presence of calcium chloride (0 mM, 20 mM and 50 mM) were followed by combining electrochemical determination of free calcium with the determination of soluble calcium by inductively coupled plasma optical emission spectroscopy. α-Lactalbumin and, more evident, ß-lactoglobulin were found to increase calcium bioaccessibility with increasing intestinal digestion time by around 5% and 10%, respectively, due to the complex binding of calcium to peptides formed from protein hydrolysis by gastrointestinal enzymes. In vitro digested samples of ß-lactoglobulin in presence of CaCl2 had nearly twice as much complex bound calcium as α-lactalbumin samples. The calcium bioaccessibility decreased significantly with the increasing concentration of added calcium chloride, although the amount of calcium chloride had little effect on the extension of digestion of α-lactalbumin and ß-lactoglobulin. Simulated digestion fluids were found to have a negative effect on calcium bioaccessibility, especially the presence of hydrogen phosphate, and the amount of precipitated calcium increased significantly with increasing amount of added calcium chloride. Based on analysis and visualization by sequences of the peptides formed during digestion of α-lactalbumin and ß-lactoglobulin, it was observed that peptides containing aspartic acid and glutamic acid acting as calcium chelators, may prevent precipitation of calcium in the intestines and increase calcium bioaccessibility. These results provide knowledge for the design of new dairy based functional foods to prevent calcium deficiency.

Temperature effect on calcium binding to aspartate and glutamate.

Aspartate (Asp) mononegative ion binds calcium through both carboxylates in contrast to binding through only the side chain carboxylate for mononegative glutamate (Glu), as shown by density functional theory (DFT) calculations. A stronger binding was confirmed electrochemically for Asp compared to Glu. From temperature dependence of binding constant, 15-37 °C investigated for aqueous 0.16 M NaCl, a more negative ΔH0 of - 21 kJ·mol-1 was found for Glu compared to ΔH0 =  -17 kJ·mol-1 for Asp, a difference confirmed by DFT calculations and qualitatively also by isothermal titration calorimetry. The stronger binding of calcium to Asp (Kass,c = 5.3 M-1 at 37 °C) compared to Glu (Kass,c = 3.6 M-1 at 37 °C) despite the less negative enthalpy of binding is accordingly an entropy effect due to ring formation in the complex for Asp.

Double-Site Binding and Anti-/Pro-oxidation of Luteolin on Bovine Serum Albumin Mediated by Copper(II) Coordination.

The interactions of luteolin (Lut) with bovine serum albumin (BSA) mediated by Cu(II) were investigated by spectroscopic, calorimetric, and molecular dynamic (MD) methods. Fluorescence studies showed that the binding of Lut to BSA was significantly enhanced by Cu(II) coordination with the number of binding sites and binding constant increasing from n = 1 and K a = 3.2 × 105 L·mol-1 for Lut to n = 2 and K a = 7.1 × 105 L·mol-1 for a 1:1 Cu(II)-luteolin complex, in agreement with the results from isothermal titration calorimetry (ITC). Site-specific experiments with warfarin and ibuprofen and MD confirmed that two binding sites of BSA were sequentially occupied by two Cu(II)-luteolin complexes. Cu(II) coordination increased the antioxidant activity of luteolin by 60% in the inhibition of carbonyl formation from the oxidation of amino groups in the side chain of BSA induced by the peroxyl radical ROO•; however, it counteracted the antioxidant effects of luteolin and played pro-oxidative roles in BSA aggregation induced by •OH.

Peroxyl radical induced membrane instability of giant unilamellar vesicles and anti-lipooxidation protection.

The present work is intended to investigate the morphological instability of lipid membrane induced by peroxyl radical (ROO•) and the underlying mechanism. To this end, the giant unilamellar vesicle (GUV) made from phosphatidylcholine was employed as a membrane model, and the azo compounds 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) and 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) were used as the precursors of ROO•. Upon mild pyrolysis, the GUV immobilized in agarose gel was followed by conventional optical microscopy in real time, and the morphological variation was quantified by the image heterogeneity, perimeter and area all as a function of time for up to an hour. Lipid oxidation initiated from lipid phase with AMVN and from aqueous phase with AAPH led to different types of morphological changes, i.e. membrane coarsening and vesicle deformation/budding, respectively. Based on the compositional analysis of lipid oxidation products, we propose that ROO• as the primary radical initiator is responsible for the morphological changes of the GUV-AMVN while both ROO• and RO• are responsible for the morphological changes of the GUV-AAPH system. Lipophilic ß-carotene and amphipathic plant phenols as antioxidants are found to be able to stabilize the membrane integrity effectively, in corroboration with the proposed mechanisms for membrane destruction.

Temperature effects on calcium binding to caseins.

The kinetics of binding of calcium ions in molar excess to individual caseins and casein ingredients was studied in pH 6.4 aqueous solutions using stopped-flow absorption spectroscopy. An initial second-order reaction, faster for ß-casein than for αs-casein due to lower energy of activation (ΔEa1,ß = 8.2 kJ∙mol-1; ΔEa1,α = 18.1 kJ∙mol-1, respectively), is followed by a slower first-order reaction with similar energies of activation (ΔEa2,ß = 25.3 kJ∙mol-1 and ΔEa2,α = 20.7 kJ∙mol-1) as determined from temperature dependence of rate between 25 °C and 50 °C. Sodium caseinate reacts faster with calcium than both αs-casein and ß-casein in the first reaction of the two consecutive reactions, while the rate of the second falls between αs-casein and ß-casein. Global spectral analysis showed the UV-visible spectra of the reaction intermediates of the caseins to be more similar to the final products than to the initial casein reactants. Dynamic and static light scattering indicated decreasing particle sizes and increasing particle surface upon calcium-binding most significantly at low temperatures. The calcium binding to casein was found endothermic by isothermal titration calorimetry. Calcium binding seems to be controlled by enthalpy/entropy compensation corresponding to an isoequilibrium temperature of 38 °C in agreement with binding of calcium to o-phosphoserine rather than to aspartate or glutamate side chains of the caseins. Binding capacity and affinity for calcium to αs-casein and sodium caseinate both increased with increasing temperature in agreement with the endothermic nature of the binding. Decreasing enthalpy of binding for each calcium indicating a decrease in heat capacity of the caseins upon calcium-binding. The small difference between binding enthalpy and energy of activation for association of calcium to αs-casein lead to the conclusion that calcium dissociation goes through an early transition state. The rate of calcium dissociation hardly depends on temperature also explaining why calcium binding to caseins is important for calcium bioaccessibility.

Increasing calcium phosphate aqueous solubility and spontaneous supersaturation combining citrate and gluconate with perspectives for functional foods.

Uptake of calcium from food depends on solubility of calcium salts in the intestines, and precipitation of calcium phosphates decreases bioaccessibility of food calcium. Citrate as a high affinity complex binder for calcium was found spontaneously to create strongly supersaturated solutions by rapid dissolution of calcium hydrogen phosphate characterized by short lag phases for precipitation. Gluconate with weaker affinity for calcium binding showed longer lag phases for precipitation from supersaturated solutions. For citrate/gluconate combinations, the highest degree of supersaturation with longest lag phases for precipitation were found by trial-and-error experiments for a citrate/gluconate ratio of 1:10 for dissolution of calcium hydrogen phosphate resulting in supersaturation factors around three and without precipitation for more than a month. The aim of the present study was to provide a physicochemical explanation of this robust supersaturation. Calcium speciation based on electrochemical calcium activity measurement identified a low [Ca2+]·[HCitr2-] product as critical for supersaturation.

Strontium increasing calcium accessibility from calcium citrate.

Strontium chloride added to aqueous suspensions of metastable calcium citrate tetrahydrate increased calcium ion activity measured electrochemically without transition of metastable tetrahydrate to stable calcium citrate hexahydrate as shown by DSC. Calcium activity increase was explained by lower solubility of strontium citrate pentahydrate formed (8.9 × 10-4 M at 25 °C) increasing with temperature compared to calcium citrate tetrahydrate (1.6 × 10-3 M) decreasing with temperature. Strontium binding to citrate was found endothermic, ΔH0 = 45 kJ∙mol-1 at 25 °C, while calcium binding shows variation from ΔH0 = 94 kJ∙mol-1 at 10 °C becoming exothermic above physiological temperature with ΔH0 = -9 kJ∙mol-1 at 45 °C as determined from temperature and concentration variation in electric conductivity. These differences in solution thermodynamics and pH effect on complex formation between calcium and strontium citrate are discussed in relation to biomineralization.

Enthalpy-entropy compensation in calcium binding to acid-base forms of glycine tyrosine dipeptides from hydrolysis of α-lactalbumin.

Calcium binding to peptides formed by hydrolysis of whey proteins during digestion is important for calcium uptake in the intestines and affects the antioxidant function of the peptides. For the two dipeptides, Gly-Tyr and Tyr-Gly, potential hydrolysis products of α-lactalbumin, calcium binding to the three forms of each dipeptide in acid-base equilibrium at intestinal pH was determined electrochemically and compared to binding to tyrosine for aqueous 0.16 M NaCl for 5 < pH < 9 at 15 °C, 25 °C, and 37 °C. At milk pH at 25 °C, binding of calcium to the zwitterion of GlyTyr dominates, with an association constant Kass2 = 22 M-1 with ΔH0 = -46 kJ·mol-1, while binding to the mononegative TyrGly dominates for TyrGly with Kass3 = 32 M-1 and ΔH0 = -38 kJ·mol-1. At intestinal conditions, pH = 7 and 37 °C, binding of calcium has similar affinity for GlyTyr and TyrGly, while at higher pH and lower temperature, GlyTyr binds stronger. Density Functional Theory calculations confirmed a stronger binding to the zwitterion of GlyTyr than of TyrGly and an increasing affinity with increasing pH for both. Calcium binding to the acid/base forms of the dipeptides is at neutral pH strongly exothermic with ΔH0 becoming less negative at higher pH, and a linear enthalpy-entropy compensation (r2 = 0.99) results in comparable binding important for calcium bioavailability along the changing distribution among acid-base forms. Calcium binding decreases radical scavenging rate and antioxidative activity of both dipeptides.

Spatial effects of photosensitization on morphology of giant unilamellar vesicles.

Singlet oxygen (1O2) formed through photosensitization may initiate oxidative destruction of biomembranes, however, the influence from the spatial organization of photosensitizers (PS) relative to membranes remains unclear. To clarify this issue, we loaded riboflavin 5'-(dihydrogen phosphate) monosodium (FMN-Na) as a hydrophilic PS into the lumen of halloysite nanotubes (HNTs), and attached the nanoassemblies (FMN-Na@HNTs), via Pickering effects, to the outer surfaces of giant unilamellar vesicles (GUVs) of phospholipids. We also prepared GUVs dopped with lumiflavin (LF) as a lipophilic PS having a 1O2 quantum yield comparable to FMN-Na. FMN-Na capsulated in HNT was characterized by a longer triplet excited state lifetime (12.1 µs) compared to FMN-Na free in solution (7.5 µs), and FMN-Na in both forms efficiently generated 1O2 upon illumination. The spatio-effects of PS on the photosensitized morphological changes of membranes were studied using conventional optical microscopy by monitoring GUV morphological changes. Upon light exposure (400-440 nm), the GUVs attached with FMN-Na@HNT merely experienced membrane deformation starting from the original spherical shape, ascribed to Type II photosensitization with 1O2 as oxidant. In contrast, photooxidation of LF dopped GUVs mainly led to membrane coarsening and budding assigned to Type I photosensitization. The spatial effects of PS on photosensitized morphological changes were related to the different lipid oxidation products generated through Type I and Type II photosensitized lipid oxidation.

Hydrates of calcium citrate and their interconversion in relation to calcium bioaccessibility.

Calcium citrate tetrahydrate (CCT) and hexahydrate (CCH) precipitates from aqueous solutions of CaCl2 and sodium citrate above and below the transition temperature of 52 °C, respectively. The CCT, the dihydrate (CCD) and anhydrate (CCA) as obtained by a stepwise dehydration of solid CCH have enthalpy of dehydration of ΔH0CCH to CCT = 43.6, ΔH0CCT to CCD = 43.8, and ΔH0CCD to CCA = 88.1 kJ∙mol-1 as measured by DSC. WAXS measurements demonstrate a stepwise decrease in unit cell size upon dehydration, and a stronger binding of the two first water compared to additional. The increasing negative enthalpy of dissolution, as calculated from the temperature dependence of solubility (10-90 °C), +21 kJ∙mol-1 (CCH), -20 kJ∙mol-1 (CCT), -22 kJ∙mol-1 (CCD), and -40 kJ∙mol-1 (CCA) shows along the series of hydrates with increasing solubility, enthalpy-entropy compensation with an isoequilibrium temperature of 49 °C. Conversion of CCD and CCA in aqueous solutions yields the more soluble CCT, not the stable CCH in agreement with Ostwald's stage law, increasing calcium bioaccessibility under physiological conditions in intestines.

Promotion effects of flavonoids on browning induced by enzymatic oxidation of tyrosinase: structure-activity relationship.

Tyrosinase, widely distributed in nature, is a copper-containing polyphenol oxidase involved in the formation of melanin. Flavonoids are most often considered as tyrosinase inhibitors but have also been confirmed to be tyrosinase substrates. Four structure-related flavonoids including flavones (apigenin and luteolin) and flavonols (kaempferol and quercetin) are found to promote not inhibit browning induced by tyrosinase catalyzed oxidation both in model systems and in mushrooms under aerobic conditions. A comparison with enzymatic oxidation and autooxidation of flavonoids alone has helped to clarify why flavonoids function as a substrate rather than an inhibitor. Flavonoids almost do not affect the kinetics of melanin formation from enzymatic oxidation of l-dopa in excess. In addition, a new brown complex formed during the reaction of flavonoid quinone and dopaquinone is suggested to enhance the browning effects by competing with isomerization and autooxidation. Structure-activity relationships of the four flavonoids in melanin formation leading to browning induced by autooxidation and enzymatic oxidation confirm the enzymatic nature of the browning.

ESR spin trapping for in situ detection of radicals involved in the early stages of lipid oxidation of dried microencapsulated oils.

Different studies have shown that detection of free radicals by ESR spin trapping provides useful information on the susceptibility to oxidation of bulk oils and accordingly on the oxidative stability of different samples for comparative purposes. With the same goal, ESR spin trapping was evaluated in this work for in situ detection of radicals in dried microencapsulated oils (DMOs). By testing different oils, encapsulation matrices and oxidation conditions, results showed that ESR spin trapping can be useful to evaluate the oxidative susceptibility of DMOs, but ESR data should be interpreted cautiously, as the great complexity of the reactions involved may lead to data misinterpretations. Conditions for oxygen availability can have important impacts on the rates of both spin trapping and spin-adduct quenching affecting the levels of radicals observed. The kinetics of oxidation, spin trapping and spin-adduct decay should be known first in bulk oils for correct data interpretation in DMOs.

Lime Juice Enhances Calcium Bioaccessibility from Yogurt Snacks Formulated with Whey Minerals and Proteins.

Yogurt-based snacks originally with a calcium content between 0.10 and 0.17 mmol/g dry matter were enriched with a whey mineral concentrate and whey protein isolate or hydrolysate. Whey mineral concentrate was added to increase the total amount of calcium by 0.030 mmol/g dry matter. Calcium bioaccessibility was determined following an in vitro protocol including oral, gastric, and intestinal digestion, with special focus on the effect of lime juice quantifying calcium concentration and activity. Calcium bioaccessibility, defined as soluble calcium divided by total calcium after intestinal digestion amounted to between 17 and 25% for snacks without lime juice. For snacks with lime juice, the bioaccessibility increased to between 24 and 40%, an effect attributed to the presence of citric acid. Citric acid increased the calcium solubility both from whey mineral concentrate and yogurt, and the citrate anion kept supersaturated calcium soluble in the chyme. The binding of calcium in the chyme from snacks with or without lime juice was compared electrochemically, showing that citrate increased the amount of bound calcium but with lower affinity. The results indicated that whey minerals, a waste from cheese production, may be utilized in snacks enhancing calcium bioaccessibility when combined with lime juice.

Calcium availability from whey mineral residues increased by hydrogen citrate.

Insoluble mineral residues from whey processing dominated by hydroxyapatite and calcium hydrogen phosphate were found to dissolve isothermally in aqueous sodium hydrogen citrate. Dissolution occurred spontaneously and the resultant homogeneous solutions were found to be supersaturated solutions in both calcium citrate and calcium hydrogen phosphate. Supersaturation was investigated by visual inspection combined with turbidity measurements and analyses of calcium and phosphorous by ICP. The maximal supersaturation was found to be proportional to total hydrogen citrate concentration. For 0.2 M hydrogen citrate, maximum calcium concentration was achieved in the first hours of dissolution resulting in the supersaturation of calcium hydrogen phosphate with a factor of 10. Calcium citrate rather than calcium hydrogen phosphate precipitated from the supersaturated solutions and the time elapsing before precipitation began, increased with increasing concentrations of excess of hydrogen citrate. This lag phase for precipitation ranged from several hours for 0.2 M hydrogen citrate to more than a day for higher hydrogen citrate concentrations, for which the solutions were saturated in calcium hydrogen phosphate and became supersaturated only in calcium citrate due to the strong binding of calcium by citrate. The appearance and decay of supersaturation was kinetically studied in order to provide the background for future exploration of whey minerals in functional foods for improved calcium nutrition.

Slow lactate gluconate exchange in calcium complexes during precipitation from supersaturated aqueous solutions.

Saturated solutions of calcium l-lactate in water or in deuterium oxide continuously dissolve calcium l-lactate by addition of solid sodium d-gluconate and become strongly supersaturated in calcium d-gluconate due to no or slow precipitation. The quantification of total dissolved calcium allied with the calcium complexes equilibrium constants allowed an ion speciation, which shows an initial non-thermal and spontaneous supersaturation of more than a factor of 50 at 25 °C only slowly decreasing after initiation of precipitation of calcium d-gluconate after a lag phase of several hours. A mathematical model is proposed, based on numerical solution of coupled differential equations of dynamics of l-lactate and d-gluconate exchange during the lag phase for precipitation and during precipitation. A slow exchange of l-lactate coordinated to calcium with d-gluconate is indicated with a time constant of 0.20 h-1 in water and of 0.15 h-1 in deuterium oxide and a kinetic deuterium/hydrogen isotope effect of 1.25. Such spontaneous non-thermal supersaturation and slow ligand exchange with a pseudo first order equilibration process with a half-life of 3.5 h in water for calcium hydroxycarboxylates can help to understand the higher calcium bioavailability from calcium hydroxycarboxylates compared to simple salts.

Hydroxycarboxylate combinations for increasing solubility and robustness of supersaturated solutions of whey mineral residues.

Calcium phosphates present in whey mineral residue is a potential source of calcium for dietary purposes. Combinations of aqueous isocitrate and citrate were found more efficient than each of the isomers in dissolving dried insoluble whey processing mineral residues spontaneously forming supersaturated solutions. Hydrogen isocitrate was found around 30% less efficient in these non thermal dissolution processes compared to hydrogen citrate based on amount of dissolved calcium. In contrast, the lag phase of up to 4 h for precipitation of calcium citrate from the supersaturated solutions was significantly longer when calcium isocitrate was present. Highest degree of supersaturation with longest lag phase for precipitation was found for citrate/isocitrate combinations in a 1:1 ratio. Addition of calcium saccharate during dissolution further prolonged the lag phase simultaneously preserving the higher supersaturation degrees. Combinations of the three hydroxycarboxylates seem accordingly to provide a basis for increasing calcium availability from dried whey mineral fractions consisting mainly of calcium hydrogen phosphate and hydroxyapatite of low solubility with the perspective of transforming a side stream from cheese production into valuable functional foods.

Conjugation Length Dependence of Free Radical Scavenging Efficiency of Retinal and Retinylisoflavonoid Homologues.

Retinal (C20) and the C25 and C30 homologues were compared as radical scavengers together with their C22, C27, and C32 homologues linked with daidzein through a B'3 (isoflavonoid) to oxo-carbon (aldehyde) covalent bond. Oxidation potential in acetonitrile determined by cyclic voltammetry and ionization potential calculated by density functional theory for the aldehydes and dyads (conjugates), of which the two longer are new, decreased linearly with the wavenumber for absorption maximum. The logarithm of the second-order rate constant for scavenging of the ABTS•+ increased linearly with decreasing oxidation potential suggesting that longer conjugation in the antioxidant increases the rate of electron transfer. A similar linear free energy relationship was found for the rate of scavenging DPPH•, including daidzein, which may indicate involvement of hydrogen atom transfer from an isoflavonoid phenol. Prediction of radical scavenging efficiency from visible absorption spectra was demonstrated with the perspective of rational design of bifunctional amphiphilic antioxidants.

Generation of Aggregates of α-Lactalbumin by UV-B Light Exposure.

Whey proteins are widely used as ingredients in the form of aggregates to obtain certain functionalities in food applications. The aim of this study was to understand how UV illumination generates aggregates of α-lactalbumin (α-LA) as an alternative to heat treatments traditionally used for industrial production of protein aggregates. Absorption of UV light by α-LA caused cleavage of disulfide bonds and release of thiol groups, which resulted in primarily disulfide-mediated aggregation. This process mediated efficient aggregation with up to 98% monomer conversion into aggregates through formation of intermolecular disulfide bonds, while only minor levels of nonreducible cross-links were observed. SDS-PAGE analysis revealed that illumination led to formation of dimeric, trimeric, and oligomeric forms of α-LA. LC-MS/MS analysis showed that all of the four native disulfide bonds in α-LA were cleaved by UV illumination but to different extents, and the extent of cleavage was found to be higher in the absence of calcium. Seventeen different non-native disulfides were formed after 24 h of UV illumination. Two dityrosine bonds were identified (Tyr103-Tyr103 and Tyr36-Tyr103) alongside ditryptophan (Trp118-Trp118) and tyrosine-tryptophan (Tyr50-Trp60) cross-links. In addition, Trp60, Trp118, Cys73, Cys91, Cys120, Phe80, Met90, His68, and His107 were found to be oxidized up to 12% as compared to a nonilluminated control. Our work illustrates that light exposure can be used for generation of α-LA aggregates, but optimization of the illumination conditions is required to reduce oxidative damage to Trp, Cys, Phe, Met, and His residues.

Cleavage of Disulfide Bonds in Cystine by UV-B Illumination Mediated by Tryptophan or Tyrosine as Photosensitizers.

Photolytic cleavage of disulfide bonds in proteins by UV light will influence their structure and functionality. The present study aimed to investigate the efficiency of disulfide cleavage by UV-B light in a system without a protein backbone consisting of combinations of cystine (a disulfide) and tryptophan (Trp) or tyrosine (Tyr) under anaerobic and aerobic conditions and to identify oxidation products formed by UV-B light. Cystine was reduced to cysteine (Cys) almost with a 1:1 stoichiometry by photoexcited Trp for anaerobic equimolar aqueous solutions (each 200 µM; pH 7.0), while photoexcited Tyr provided lower concentrations of Cys. The calculation of apparent quantum yields allowed for a comparison between the efficiency of reactions and showed that formation of Cys from disulfide cleavage of cystine was more efficient by photoexcited Trp than by photoexcited Tyr and of cystine alone and that Trp was more sensitive to photodegradation than Tyr and cystine under both aerobic and anaerobic conditions. Increasing the ratio between cystine and Trp to a 1:2 ratio did not increase the efficiency of free thiol formation but caused a more efficient photodegradation of Trp. The free thiol formed from disulfide cleavage of cystine was further oxidized to other unidentified compounds. Trp oxidation products (3-hydroxykynurenine (3-OH-Kyn) and tryptamine) were only identified in minor concentrations following light exposure of cystine and Trp in 1:1 and 1:2 ratios under both aerobic and anaerobic conditions, indicating further photodegradation to unidentified compounds. 3,4-Dihydroxyphenylalanine (DOPA) was formed from the oxidation of Tyr in the illuminated samples of cystine and Tyr in a 1:1 ratio under both aerobic and anaerobic conditions.