Dhayal Surender Kumar, Sforza Stefano, Wierenga Peter A, Gruppen Harry
Laboratory of Food Chemistry, Wageningen University, 6700 AA Wageningen, Wageningen, The Netherlands.
Laboratory of Food Chemistry, Wageningen University, 6700 AA Wageningen, Wageningen, The Netherlands; Department of Food Science, University of Parma, Italy.
Biochim Biophys Acta. 2015 Dec;1854(12):1898-1905. doi: 10.1016/j.bbapap.2015.08.001. Epub 2015 Aug 15.
Horseradish peroxidase (HRP) induced cross-linking of proteins has been reported to proceed through formation of di-tyrosine cross-links. In the case of low molar mass phenolic substrates, the enzymatic oxidation is reported to lead to polymerization of the phenols. The aim of this work was to investigate if during oxidative cross-linking of proteins oligo-tyrosine cross-links are formed in addition to dityrosine. To this end, α-lactalbumin (α-LA) was cross-linked using horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂). The reaction products were acid hydrolysed, after which the cross-linked amino acids were investigated by LC-MS and MALDI-MS. To test the effect of the size of the substrate, the cross-linking reaction was also performed with L-tyrosine, N-acetyl L-tyrosinamide and angiotensin. These products were analyzed by LC-MS directly, as well as after acid hydrolysis. In the acid hydrolysates of all samples oligo-tyrosine (Yn, n=3-8) was found in addition to di-tyrosine (Y2). Two stages of cross-linking of α-LA were identified: a) 1-2 cross-links were formed per monomer until the monomers were converted into oligomers, and b) subsequent cross-linking of oligomers formed in the first stage to form nanoparticles containing 3-4 cross-links per monomer. The transition from first stage to the second stage coincided with the point where di-tyrosine started to decrease and more oligo-tyrosines were formed. In conclusion, extensive polymerization of α-LA using HRP via oligo-tyrosine cross-links is possible, as is the case for low molar mass tyrosine containing substrates.
据报道，辣根过氧化物酶（HRP）诱导的蛋白质交联是通过二酪氨酸交联的形成来进行的。对于低摩尔质量的酚类底物，据报道酶促氧化会导致酚类聚合。这项工作的目的是研究在蛋白质的氧化交联过程中，除了二酪氨酸之外是否还会形成寡聚酪氨酸交联。为此，使用辣根过氧化物酶（HRP）和过氧化氢（H₂O₂）对α-乳白蛋白（α-LA）进行交联。对反应产物进行酸水解，然后通过液相色谱-质谱联用（LC-MS）和基质辅助激光解吸电离质谱（MALDI-MS）研究交联氨基酸。为了测试底物大小的影响，还使用L-酪氨酸、N-乙酰-L-酪氨酸酰胺和血管紧张素进行交联反应。这些产物直接通过LC-MS分析，以及在酸水解后进行分析。在所有样品的酸水解产物中，除了二酪氨酸（Y2）之外还发现了寡聚酪氨酸（Yn，n = 3 - 8）。确定了α-LA交联的两个阶段：a）每个单体形成1 - 2个交联，直到单体转化为寡聚物；b）第一阶段形成的寡聚物随后交联形成每个单体含有3 - 4个交联的纳米颗粒。从第一阶段到第二阶段的转变与二酪氨酸开始减少且形成更多寡聚酪氨酸的点一致。总之，与含低摩尔质量酪氨酸的底物情况一样，使用HRP通过寡聚酪氨酸交联对α-LA进行广泛聚合是可能的。
