Development of Monoclonal Antibodies Against a Riboflavin‐Tryptophan Photoinduced Adduct: Reactivity to Eye Lens Proteins*

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Time-resolved photolysis studies of riboflavin (RF) were carried out in the presence and absence of a-, b H -and b Lcrystallins of bovine eye lens. The transient absorption spectra, recorded 5 ms after the laser pulse, reveal the presence of the absorption band (625-675 nm) of the RF neutral triplet state (t 5 42 ms) accompanied by the appearance of a long-lived absorption (t 5 320 ms) in the 500-600 nm region due to the formation of the semireduced RF radical. The RF excited state is quenched by the crystallin proteins through a mechanism that involves electron transfer from the proteins to the flavin, as shown by the decrease of the triplet RF band with the concomitant increase of the band of its semireduced form. Tryptophan loss on RF-sensitized photooxidation of the crystallins when irradiated with monochromatic visible light (450 nm) in a 5% oxygen atmosphere was studied. A direct correlation was found between the triplet RF quenching rate constants by the different crystallin fractions and the decomposition rate constants for the exposed and partially buried tryptophans in the proteins. The RFsensitized photooxidation of the crystallins is accompanied by the decrease of the low molecular weight constituents giving rise to its multimeric forms. A direct correlation was observed between the initial rate of decrease of the low molecular weight bands corresponding to the irradiated a-, b H -and b Lcrystallins and the quenching constant values of triplet RF by the different crystallins. The correlations found in this study confirm the importance of the Type-I photosensitizing mechanism of the crystallins, when RF acts as a sensitizer at low oxygen concentration, as can occur in the eye lens.

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Study of the Interaction Between Triplet Riboflavin and the α-, βH- and βL-Crystallins of the Eye Lens ¶

Viteri

Edwards

Fuente

et al. 2007

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“…In this case the damage would accumulate during the whole life because of the known lack of turnover of lens proteins. These types of processes could explain the evidence of an accumulation of indole-RF photoadduct during lens aging and cataract formation (37).…”

Section: Resultsmentioning

confidence: 99%

Riboflavin Photodegradation and Photosensitizing Effects are Highly Dependent on Oxygen and Ascorbate Concentrations¶

Rochette

Silva

Birlouez‐Aragon

et al. 2007

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Riboflavin (RF) is a normal component of the eye lens which triggers a strong photosensitizing activity when exposed to light. Upon irradiation with short wavelength radiations below 400 nm, RF‐photosensitized damage may occur. However, vitamin C is present at high concentrations in the normal lens and plays an important role in inhibiting these photosensitization processes. An in vitro simple model was used with the objective of understanding better the relationships between vitamin C and oxygen concentrations on the mechanisms of RF‐mediated photodegradation of tryptophan (Trp), a target particularly sensitive to photo‐oxidation. Under nitrogen, the RF decomposition reached its maximal value, and vitamin C and Trp photo‐oxidation was negligible. When increasing oxygen pressure, RF photodegradation dropped and vitamin C photo‐oxidation strongly increased and was maximal at 100% O2. RF‐induced photodegradation of Trp first increased with oxygen concentration, up to 40 μM O2, and then decreased. RF and Trp degradation were significantly protected by vitamin C so that no more than 20% of the substrates concentration were oxidized in the presence of vitamin C higher than 0.8 mM. From our results we conclude that in the specific conditions of the normal lens, the high vitamin C concentration (2 mM) is compatible with the UVA radiation hazard, despite the presence of RF. However, if lenticular vitamin C decreases below 0.8 mM, photodegradation of RF may occur and Trp may therefore be photo‐oxidized by a Type‐I mechanism.

“…In spite of their importance for living organisms, the presence of RF and Trp in solutions of parenteral nutrition and in cell culture media exposed to visible light has been related with hepatotoxic (1–3) and cytotoxic (4–6) effects, respectively. Flavin‐sensitized photoprocesses in the eye lens have also been mentioned as one of the causes of protein aggregations during aging and cataractogenesis (7–9). Both Trp and RF are photoreactive compounds, the former as an UVB light–sensitive substrate and target of reactive oxygen species and the latter as a visible light absorber and photosensitizer, initiating both Type‐I and Type‐II photosensitized oxidations (10–13).…”

Section: Introductionmentioning

confidence: 99%

Protective Effect of Boldo and Tea Infusions on the Visible Light-mediated Pro-oxidant Effects of Vitamin B2, Riboflavin¶

Silva¹,

Jopia²,

Edwards³

et al. 2002

The effect of Boldo and black tea infusions on the pro-oxidant effects of vitamin B2, riboflavin (RF), when exposed to the action of visible light was studied. The amounts of antioxidants present in Boldo and tea infusions were evaluated by a procedure based on the bleaching of preformed 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cations and were expressed as 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid equivalent concentrations. The quenching rate constants of singlet oxygen (1O2; [kq]Boldo = 6.0 x 10(7) M(-1) s(-1) and [kq]Tea = 3.2 x 10(7) M(-1) s(-1)) and triplet RF (3RF; [3RFkq]Boldo = 10 x 10(8) M(-1) s(-1) and [3RFkq]TEA = 3.2 x 10(8) M(-1) s(-1)) with Boldo and tea were determined by flash photolysis. These data allow a quantitative interpretation of the results obtained. Our data suggest that most of the oxygen consumption observed in the photolysis of RF in the presence of tea and Boldo infusions is caused by 1O2 reactions. The oxygen consumption quantum yield is considerably smaller than the fraction of RF triplets trapped by the additives (AH) present in the infusion, indicating that their interaction with 3RF does not lead to chemical reactions or that the AH*+ radical ions initially formed participate in secondary processes that do not consume oxygen. Boldo and tea infusions have a significant protective effect when a system containing RF and tryptophan (Trp) is exposed to visible light, not only by quenching the 1O2 and interfering with the Type-I mechanism but also by repairing the damage to Trp molecules associated with the latter mechanism.