IPROCAR Research Institute, Faculty of Veterinary, University of Extremadura, 10003, Cáceres, Spain.
IPROCAR Research Institute, Faculty of Veterinary, University of Extremadura, 10003, Cáceres, Spain.
Redox Biol. 2019 Sep;26:101277. doi: 10.1016/j.redox.2019.101277. Epub 2019 Jul 20.
Carbonylation is one of the most remarkable expressions of the oxidative damage to proteins and the DNPH method the most common procedure to assess protein oxidation in biological samples. The present study was elicited by two hypotheses: i) is malondialdehyde, as a reactive dicarbonyl, able to induce the formation of allysine through a Maillard-type reaction? and ii) to which extent does the attachment of MDA to proteins interfere in the assessment of protein carbonyls using the DNPH method? Human serum albumin (HSA), human hemoglobin (HEM) and β-lactoglobulin (LAC) (5 mg/mL) were incubated with MDA (0.25 mM) for 24 h at 37 °C (HSA and HEM) or 80 °C (LAC). Results showed that MDA was unable to induce oxidative deamination of lysine residues and instead, formed stable and fluorescent adducts with proteins. Such adducts were tagged by the DNPH method, accounting for most of the protein hydrazones quantified. This interfering effect was observed in a wide range of MDA concentrations (0.05-1 mM). Being aware of its limitations, protein scientists should accurately interpret results from the DNPH method, and apply, when required, other methodologies such as chromatographic methods to detect specific primary oxidation products such as allysine.
羰基化是蛋白质氧化损伤最显著的表现之一，而二硝基苯肼（DNPH）法是评估生物样品中蛋白质氧化的最常用方法。本研究基于两个假设：i）丙二醛（MDA）作为一种反应性二羰基化合物，是否能够通过美拉德反应诱导赖氨酸的形成？ii）MDA 与蛋白质的结合在多大程度上干扰了使用 DNPH 方法评估蛋白质羰基？人血清白蛋白（HSA）、人血红蛋白（HEM）和β-乳球蛋白（LAC）（5mg/mL）与 MDA（0.25mM）在 37°C（HSA 和 HEM）或 80°C（LAC）下孵育 24 小时。结果表明，MDA 不能诱导赖氨酸残基的氧化脱氨，而是与蛋白质形成稳定且具有荧光的加合物。这些加合物可以通过 DNPH 方法标记，占定量的大多数蛋白质腙。这种干扰效应在广泛的 MDA 浓度（0.05-1mM）下都观察到。鉴于其局限性，蛋白质科学家应该准确解释 DNPH 方法的结果，并在需要时应用其他方法，如色谱方法，以检测特定的初级氧化产物，如丙醛赖氨酸。
