Fluorescence labeling of carbonylated lipids and proteins in cells using coumarin-hydrazide

Abstract

Carbonylation is a generic term which refers to reactive carbonyl groups present in biomolecules due to oxidative reactions induced by reactive oxygen species. Carbonylated proteins, lipids and nucleic acids have been intensively studied and often associated with onset or progression of oxidative stress related disorders. In order to reveal underlying carbonylation pathways and biological relevance, it is crucial to study their intracellular formation and spatial distribution. Carbonylated species are usually identified and quantified in cell lysates and body fluids after derivatization using specific chemical probes. However, spatial cellular and tissue distribution have been less often investigated. Here, we report coumarin-hydrazide, a fluorescent chemical probe for time- and cost-efficient labeling of cellular carbonyls followed by fluorescence microscopy to evaluate their intracellular formation both in time and space. The specificity of coumarin-hydrazide was confirmed in time- and dose-dependent experiments using human primary fibroblasts stressed with paraquat and compared with conventional DNPH-based immunocytochemistry. Both techniques stained carbonylated species accumulated in cytoplasm with strong perinuclear clustering. Using a complimentary array of analytical methods specificity of coumarin-hydrazide probe towards both protein- and lipid-bound carbonyls has been shown. Additionally, co-distribution of carbonylated species and oxidized phospholipids was demonstrated.

Cross-talk between lipid and protein carbonylation in a dynamic cardiomyocyte model of mild nitroxidative stress

“…In agreement with previous reports [33], [54], [55], carbonylated species were not equally distributed over the whole cell but displayed strong perinuclear accumulation. Slight accumulation of carbonyl specific fluorescence signal around the cell nucleus was detected even in untreated cells.…”

“…In contrast to the commonly used immunochemical detection, CHH derivatization not only allows detection of carbonylated proteins but also lipids [33]. Thus, the rapid increase in cellular carbonylation observed 15 min after SIN-1 treatment can be attributed to at least two types of biomolecules.…”

“…To assess cellular nitroxidative stress upon SIN-1 treatment, total biomolecule carbonylation was monitored using the specific membrane permeable fluorescent probe 7-(diethylamino)-coumarin-3-carbohydrazide (CHH) (Fig. 1A) [33], [53]. A significant increase in CHH fluorescence (175%) was observed already 15 min after SIN-1 addition indicating rapid oxidation of biomolecules by ROS/RNS.…”

A Highly Sensitive Fluorogenic Assay for the Detection of Nephrotoxin-Induced Oxidative Stress in Live Cells and Renal Tissue

“…Carbonylation is commonly detected using alpha-effect amines as reporter molecules in biochemical assays [7,8]. The conventional assays often require lengthy tedious downstream processing and harsh chemical components that can alter subcellular structures, thereby misrepresenting spatial distribution of carbonylated biomolecules [9]. Moreover, end-point analysis of fixed cells or cell lysates was the only option until we demonstrated the first live cell compatible assay using synthetic probes, coumarin hydrazine (CH) and benzocoumarin hydrazine (BzCH) [10,11].…”

“…Moreover, end-point analysis of fixed cells or cell lysates was the only option until we demonstrated the first live cell compatible assay using synthetic probes, coumarin hydrazine (CH) and benzocoumarin hydrazine (BzCH) [10,11]. Our approach was also validated by Vemula et al using a commercially available probe, DCCH [9]. Since crucial prerequisites for identifying mild phenotypes of chemical toxicity are high sensitivity and versatility of the assay, this work is aimed at achieving these objectives.…”