Gammelgaard Simon K, Petersen Steffen B, Haselmann Kim F, Nielsen Peter Kresten
Global Research Technologies, Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark.
Department of Health Science and Technology, Aalborg University, Fredrik Bajers Vej 7, 9220 Aalborg, Denmark.
ACS Omega. 2020 Apr 3;5(14):7962-7968. doi: 10.1021/acsomega.9b04375. eCollection 2020 Apr 14.
Ultraviolet (UV) light has been shown to induce reduction of disulfide bonds in proteins in solution. The photoreduction is proposed to be a result of electron donation from excited Tyr or Trp residues. In this work, a powerful UV femtosecond laser was used to generate photoreduced products, while the hypothesis of Tyr/Trp mediation was studied with spectroscopy and mass spectrometry. With limited irradiation times of 3 min or less at 280 nm, the laser-induced reduction in arginine vasopressin and human insulin led to significant yields of ∼3% stable reduced product. The photogenerated thiols required acidic pH for stabilization, while neutral pH primarily caused scrambling and trisulfide formation. Interestingly, there was no direct evidence that Tyr/Trp mediation was a required criterion for the photoreduction of disulfide bonds. Intermolecular electron transfer remained a possibility for insulin but was ruled out for vasopressin. We propose that an additional mechanism should be increasingly considered in UV light-induced reduction of disulfide bonds in solution, in which a single UV photon is directly absorbed by the disulfide bond.
紫外线（UV）已被证明能使溶液中的蛋白质中二硫键减少。这种光还原作用被认为是激发态的酪氨酸（Tyr）或色氨酸（Trp）残基进行电子供体作用的结果。在这项工作中，使用了强大的紫外飞秒激光来生成光还原产物，同时利用光谱学和质谱学对Tyr/Trp介导的假说进行了研究。在280nm波长下照射时间限制在3分钟或更短时间时，激光诱导的精氨酸加压素和人胰岛素的还原反应产生了约3%的稳定还原产物，产率可观。光生成的硫醇需要酸性pH来稳定，而中性pH主要导致二硫键重排和三硫键形成。有趣的是，没有直接证据表明Tyr/Trp介导是二硫键光还原的必要条件。分子间电子转移对胰岛素来说仍是一种可能性，但对加压素来说则被排除。我们提出，在紫外线诱导溶液中二硫键还原的过程中，应越来越多地考虑一种额外的机制，即二硫键直接吸收单个紫外光子。
