Protein Analytical Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, California, 94080, USA.
Late Stage Pharmaceutical Development, Genentech, Inc., 1 DNA Way, South San Francisco, California, 94080, USA.
Pharm Res. 2018 Feb 20;35(3):67. doi: 10.1007/s11095-017-2339-4.
Light is known to induce histidine (His) oxidation and His-His crosslinking in proteins. The crosslinking is resulted from the nucleophilic attack of a His to a photooxidized His from another protein. The goal of this work is to understand if covalent buffer adducts on His residues can be generated by light through similar mechanisms in nucleophilic buffers such as Tris and His.
A model protein (DNase) was buffer exchanged into nucleophilic buffers before light exposure. Photogenerated products were characterized by tryptic peptide mapping with mass spectrometry (MS) analysis. Several buffer adductions on His residues were identified after light exposure. To understand the influencing factors of such reactions, the levels of adducts were measured for six nucleophilic buffers on all His residues in DNase.
The levels of adducts were found to correlate with the solvent accessibility of the His residue. The levels of adducts also correlate with the structure of the nucleophile, especially the steric restrictions of the nucleophile. The levels of adducts can be higher than that of other His photoreaction products, including photooxidation and crosslinking.
In nucleophilic buffers, light can induce covalently-linked adducts to His residues.
已知光会诱导蛋白质中组氨酸（His）氧化和 His-His 交联。交联是由另一个蛋白质中光氧化的 His 对 His 的亲核攻击引起的。这项工作的目的是了解在亲核缓冲液如 Tris 和 His 中，共价缓冲加合物是否可以通过类似的机制在光的作用下在 His 残基上生成。
在光暴露之前，将模型蛋白（DNase）缓冲交换到亲核缓冲液中。用光激发后，通过胰蛋白酶肽图与质谱（MS）分析来表征光产物。光激发后鉴定出几个 His 残基上的缓冲加合物。为了了解这些反应的影响因素，在 DNase 上的所有 His 残基上测量了六种亲核缓冲液的加合物水平。
发现加合物的水平与 His 残基的溶剂可及性相关。加合物的水平也与亲核体的结构有关，特别是亲核体的空间位阻。加合物的水平可以高于其他 His 光反应产物，包括光氧化和交联。
在亲核缓冲液中，光可以诱导 His 残基上的共价键加合物。
