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

Abstract

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.

Effect of Addition of Tryptophan on Aggregation of Apo-α-Lactalbumin Induced by UV-Light

“…level of disulfide cleavage, or that the free thiols released from disulfide cleavage would be further oxidized [7,16]. The percentage of non-reducible covalent cross-links was not significantly different between samples added Trp illumination under aerobic and anaerobic conditions indicating that the effect of Trp and oxygen only affected disulfide-mediated aggregation and not other non-reducible cross-links.…”

“…The hydrolysates were neutralized with 4 M sodium hydroxide. The neutralized protein hydrolysates and the fraction containing free Trp obtained from centrifugal filtration were independently spiked with 100 μL of 50 μg/μL internal standard, 5-methyl- dl -Trp, and filtered through a 0.2 µm filter before injection (5 µL) onto a UHPLC system equipped with a Syncronis aQ C18 column (2.1 ID × 100 mm length, 1.7 µm particles) (Thermo Fisher Scientific, Torrance, CA, USA), as described by Zhao et al [ 16 ].…”

“…This leads to excited states Trp and Tyr and release of solvated electrons [ 14 , 15 ], which have been shown to cleave the disulfide bonds in α-LA and release free thiols [ 11 , 13 ]. In a previous study, we have shown that it is possible to achieve an efficient cleavage of disulfide bonds and release of free thiols (close to 50%) in an aqueous solution of cystine and Trp under anaerobic conditions after UV light treatment [ 16 ]. The apparent quantum yields of free thiol formation obtained in the study with free amino acids showed that the release of free thiols was more efficient under anaerobic conditions.…”

“…The apparent quantum yields of free thiol formation obtained in the study with free amino acids showed that the release of free thiols was more efficient under anaerobic conditions. In addition, the cleavage of disulfide bonds was more efficient when cystine was irradiated in the presence of Trp (1:1 molar ratio) compared to Tyr (1:1 molar ratio) or cystine alone [ 16 ]. Nevertheless, increasing the molar concentration of the Trp to 2:1 ratio with respect to cystine resulted in lower quantum yield for the release of free thiols and higher quantum yields for degradation of Trp, which indicated enhanced oxidation of Trp [ 16 ].…”

“…In addition, the cleavage of disulfide bonds was more efficient when cystine was irradiated in the presence of Trp (1:1 molar ratio) compared to Tyr (1:1 molar ratio) or cystine alone [ 16 ]. Nevertheless, increasing the molar concentration of the Trp to 2:1 ratio with respect to cystine resulted in lower quantum yield for the release of free thiols and higher quantum yields for degradation of Trp, which indicated enhanced oxidation of Trp [ 16 ]. The aim of the present study was therefore to investigate the effect of addition of free Trp on UV-B-induced aggregation of apo-α-LA under both aerobic and anaerobic conditions.…”