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Protein carbonyl determination by a rhodamine B hydrazide-based fluorometric assay.

ABSTRACT: A new fluorometric assay is presented for the ultrasensitive quantification of total protein carbonyls, and is based on their specific reaction with rhodamine B hydrazide (RBH), and the production of a protein carbonyl-RBH hydrazone the fluorescence of which (at ex/em 560/585?nm) is greatly enhanced by guanidine-HCl. Compared to the fluorescein-5-thiosemicarbazide (FTC)-based fluorometric assay, the RBH assay uses a 24-fold shorter reaction incubation time (1?h) and at least 1000-fold lower protein quantity (2.5?µg), and produces very reliable data that were verified by extensive standardization experiments. The protein carbonyl group detection sensitivity limit of the RBH assay, based on its standard curve, can be as low as 0.4 pmol, and even lower. Counting the very low protein limit of the RBH assay, its cumulative and functional sensitivity is 8500- and 800-fold higher than the corresponding ones for the FTC assay. Neither heme proteins hemoglobin and cytochrome c nor DNA interfere with the RBH assay.

SUBMITTER: Georgiou CD

PROVIDER: S-EPMC6006725 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Protein carbonyl determination by a rhodamine B hydrazide-based fluorometric assay.

Georgiou Christos D CD Zisimopoulos Dimitrios D Argyropoulou Vasiliki V Kalaitzopoulou Electra E Ioannou Panayiotis V PV Salachas George G Grune Tilman T

Redox biology 20180425

A new fluorometric assay is presented for the ultrasensitive quantification of total protein carbonyls, and is based on their specific reaction with rhodamine B hydrazide (RBH), and the production of a protein carbonyl-RBH hydrazone the fluorescence of which (at ex/em 560/585 nm) is greatly enhanced by guanidine-HCl. Compared to the fluorescein-5-thiosemicarbazide (FTC)-based fluorometric assay, the RBH assay uses a 24-fold shorter reaction incubation time (1 h) and at least 1000-fold lower prot ...[more]

PMID: 29727801

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Project description:A new 2,4-dinitrophenylhydrazine (DNPH)-based photometric assay is developed for the quantification of carbonyls in protein samples from any biological source by protein carbonyl-DNPH hydrazone formation at acidic pH in the presence of denaturing urea, and subsequent hydrazone solubilization in the presence of SDS and stabilization from acid hydrolysis at pH 7.0. At this neutral (ntr) pH, interfering unreacted DNPH is uncharged and its thus increased hydrophobicity permits its 100% effective removal from the solubilizate with ethyl acetate/hexane wash. The ntrDNPH assay is more reliable and sensitive than the standard (std) DNPH photometric assay because it eliminates its main limitations: (i) interfering unreacted DNPH (pKa 1.55) that is nonspecifically bound to the TCA (pKa 0.7)-protein pellet is not effectively removed after wash with EtOH: ethyl acetate because it is positively charged, (ii) acid (TCA-induced) hydrolysis of the protein carbonyl-DNPH hydrazone, (iii) sample protein concentration re-determination, (iv) loss of sample acid (TCA)-soluble proteins, (v) DNA interference, and (vi) requires high protein quantity samples (??1?mg). Considering ntrDNPH assay's very low protein limit (1?µg), its cumulative and functional sensitivities are 2600- and 2000-fold higher than those of the stdDNPH assay, respectively. The present study elucidates the DNA interference mechanism on the stdDNPH assay, and also develops a standardized protocol for sample protein treatment and fractionation (into cytoplasmic/aqueous, membrane/lipid-bound, and histone/DNA-bound proteins; see Supplement section V) in order to ensure reproducible carbonyl determination on defined cell protein fractions, and to eliminate assay interference from protein samples containing (i) Cys sulfenic acid groups (via their neutralization with dithiothreitol), and (ii) DNA (via its removal by streptomycin sulfate precipitation). Lastly, the ntrDNPH assay determines carbonyl groups on cell wall polysaccharides, thus paving the way on studies to investigate cell walls acting as antioxidant defense in plants, fungi, bacteria and lichens.

Project description:Trehalose is a non-reducing disaccharide that is used as an osmolyte, transport sugar, carbon reserve and stress protectant in a wide range of organisms. In plants, trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is thought to be a signal of sucrose status. Trehalose itself may play a role in pathogenic and symbiotic plant-microbe interactions, in responses to abiotic stress and in developmental signalling, but its precise functions are unknown. A major obstacle to investigating its function is the technical difficulty of measuring the very low levels of trehalose usually found in plant tissues, as most of the established trehalose assays lack sufficient specificity and/or sensitivity.A kinetic assay for trehalose was established using recombinant Escherichia coli cytoplasmic trehalase (treF), which was shown to be highly specific for trehalose. Hydrolysis of trehalose to glucose is monitored fluorometrically and the trehalose content of the tissue extract is determined from an internal calibration curve. The assay is linear for 0.2-40 pmol trehalose, and recoveries of trehalose were ?88%. A. thaliana Col-0 rosettes contain about 20-30 nmol g-1FW of trehalose, increasing to about 50-60 nmol g-1FW in plants grown at 8°C. Trehalose is not correlated with sucrose content, whereas a strong correlation between Tre6P and sucrose was confirmed. The trehalose contents of ear inflorescence primordia from the maize ramosa3 mutant and wild type plants were 6.6±2.6 nmol g-1FW and 19.0±12.7 nmol g-1FW, respectively. The trehalose:Tre6P ratios in the ramosa3 and wild-type primordia were 2.43±0.85 and 6.16±3.45, respectively.The fluorometric assay is highly specific for trehalose and sensitive enough to measure the trehalose content of very small amounts of plant tissue. Chilling induced a 2-fold accumulation of trehalose in A. thaliana rosettes, but the levels were too low to make a substantial quantitative contribution to osmoregulation. Trehalose is unlikely to function as a signal of sucrose status. The abnormal inflorescence branching phenotype of the maize ramosa3 mutant might be linked to a decrease in trehalose levels in the inflorescence primordia or a downward shift in the trehalose:Tre6P ratio.

Dataset Information

Protein carbonyl determination by a rhodamine B hydrazide-based fluorometric assay.

Publications

Protein carbonyl determination by a rhodamine B hydrazide-based fluorometric assay.

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