Methods To Examine Oxidatively Carbonylated Proteins And Cell Walls

Dr. C. Georgiou’s lab is internationally recognized for its expertise in the development of analytical methods for the in vivo/vitro quantification of the various parameters of oxidative stress. His lab is also leading in the development of the new field of Oxidative Astrobiology, where it has developed methods for the identification of life-inhibiting oxidative stress-inducing oxidants (metal superoxides and peroxides, and hydroxyl radicals) in planetary soils/plums, in collaboration with NASA Ames Research Center (Space Science and Astrobiology Division, CA, USA), Search for Extraterrestrial Intelligence Institute (SETI, CA, USA), Desert Research Institute (Las Vegas, NV, USA) and University of California at Santa Cruz (Departments of Biomolecular Engineering, and Chemistry and Biochemistry).

Oxidatively carbonylated proteins are one of the most reliable indicators of oxidative stress, mainly because they are not repaired or degraded and, thus, accumulate in cells. Although moderately carbonylated proteins are degraded by the proteasomal system, the heavily carbonylated ones tend to form high-molecular-weight aggregates that escape proteasomal degradation. Instead, they accumulate in cells as damaged or unfolded proteins, which can become cytotoxic by being been associated with a large number of diseases; they cause a progressive increase in protein aggregation and cross-linking in non-dividing (post-mitotic) cells, and they may eventually induce cell apoptosis. Accumulation of such protein aggregates may be due to the fact that they exhibit structural constraints which prevent their recognition by the catalytic sites inside the cylinder of the proteasome complex.

Other reasons for the limited withdrawal of carbonylated proteins by proteasomes is that they themselves can be inhibited by these aggregates, and could become the target of carbonylation (e.g., subunit S6 ATPase) and of other oxidative modifications (e.g. glycoxidation, and modification with lipid peroxidation products), all of which end up in proteasome (e.g., the 26S proteasome) decreased activity. Another advantage of using carbonylated proteins as reliable indicators of oxidative stress is their chemical stability which allows easy detection even upon prolonged storage. Another one is the existence of numerous assays for measuring protein carbonyls, although not always reliable (not specific, semi-quantitative and not highly sensitive).

Protein carbonyls have been measured mainly by the 2,4-dinitrophenylhydrazine (DNPH)-antibody based ELISA and Western immunoblotting assays, and by the photometric standard DNPH assay (stdDNPH assay) and the fluorometric thiosemicarbazide (FTC) assay (FTC assay). The main drawbacks of the ELISA/Western techniques are that they produce variable data due to the impartial access of the DNP antibody to every carbonyl-DNPH hydrazone adduct in the protein; they are time-consuming and quite expensive (as they use antibodies); and their accuracy may also be compromised by the nonspecific binding of free DNPH to proteins.

Reproducibility, use of high protein amount, DNA interference, loss of acidic proteins are serious reliability problems for of the stdDNPH and the FTC assay, which are addressed by my lab’s two new technologies [Georgiou, C. D., Zisimopoulos, D., Argyropoulou, V., Kalaitzopoulou, E., Salachas, G., Grune, T. (2018). Protein and cell wall polysaccharide carbonyl determination by a neutral pH 2,4-dinitrophenylhydrazine-based photometric assay. Redox Biology 17: 128-142, and Georgiou, C. D., Zisimopoulos, D., Argyropoulou, V., Kalaitzopoulou, E., Ioannou, P. V., Salachas, G., Grune, T. (2018). Protein carbonyl determination by a rhodamine B hydrazide-based fluorometric assay. Redox Biology 17: 236-245]. These technologies are:

1. The photometric ntrDNPH assay (an extensive modification of the stdDNPH assay, performed at neutral, ntr, pH). It also determines, for the first time, carbonyl groups on cell wall polysaccharides; thus, paving the way for studies that investigate cell walls with an additional role, that of antioxidant barrier defense, in plants, fungi, bacteria, and lichens.

2. The fluorometric RBH assay which uses rhodamine B hydrazide (RBH), a reagent never used before on proteins. Both assays are combined with a new protocol for fractionating and treating protein samples, and their advantages over the stdDNPH and FTC assays are as follows.

The ntrDNPH assay vs the stdDNPH assay:

• The ntrDNPH assay does not require the removal of nucleic acids from the protein sample.
• It removes the unreacted DNPH with 100% efficiency by liquid extraction with organic solvents of the protein sample while keeping it water soluble at pH 7.0. The protein pellet (TCA-precipitated) extraction (at pH 0) followed by the stdDNPH assay, ends up in losing a substantial fraction (~30%) of the initial protein, part of which are the sample’s acid proteins which are lost during the assay’s TCA-precipitation step.
• The liquid extraction of unreacted DNPH at pH 7 by the ntrDNPH assay keeps the protein carbonyl-hydrazone complex stable (for at least 2 days in the dark and at RT), whereas the same complex is unstable at the pH 0 of the solid extraction of the stdDNPH assay.
• The liquid extraction of unreacted DNPH strategy of the ntrDNPH assay does not decrease the initial protein quantity used. Thus, it does not require protein redetermination (as the stdDNPH assay does) and allows its application on protein samples as low as 1 µg, which is ~1,000-fold lower than the protein quantity used by the stdDNPH assay (1-2 mg). Accounting for the inefficient removal of unreacted DNPH by the stdDNPH assay and the extremely low protein sample that can be used by the ntrDNPH assay, the cumulative sensitivity of this assay is 2,600-fold higher than that of the stdDNPH assay. The high sensitivity and the very low protein quantity of the new DNPH assay extend its applicability to any biological sample.
• It uses 10-fold lower DNPH concentration in comparison to the stdDNPH assay.
• The reaction time of the DNPH reagent with the protein carbonyls in the ntrDNPH assay is at least one half that of the stdDNPH assay.

The RBH assay vs the FTC assay:

• The RBH assay does not require the removal of nucleic acids from the protein sample.
• It uses protein samples as low as 3 µg (compared to the ≥2 mg protein samples used by the FTC assay), it recovers the protein sample with ~95% efficiency (by a combination of deoxycholic acid-TCA precipitation of the RBH-treated protein sample, instead of the ineffective TCA precipitation used by the FTC assay), and it measures also sample’s acidic proteins (which are lost during the protein TCA-precipitation step of the FTC assay).
• The reaction time of the RBH assay is 1 hr, while that of the FTC assay is 24 hrs.
• The fluorescence of the hydrazone formed between the RBH reagent and the protein carbonyls is stable for at least two days in the dark and at RT.
• The fluorescence emission of the protein carbonyl-hydrazone formed by the new fluorogenic reagent is ~100-fold higher than that of the protein carbonyl-FTC hydrazone. When combined with the minimum protein quantity required by the new assay (2.5 µg, or 800-fold lower than that of the FTC assay), this gives the RBH assay a cumulative sensitivity 8,500-fold higher than that of the FTC assay. This extremely high sensitivity extends its applicability to any biological sample if combined with the ultrasensitive assay for protein quantification (down to 100 ng) developed also by Dr. C. Georgiou’s lab [Georgiou, C. D., Grintzalis, K., Zervoudakis, G., Papapostolou, I. (2008). Mechanism of Coomassie brilliant blue G-250 binding to proteins: a hydrophobic assay for nanogram quantities of proteins. Analytical and Bioanalytical Chemistry 391: 391-403].
• The fluorogenic reagent (RBH) used by the RBH assay is quite inexpensive (compared to the very costly FTC reagent), can be easily synthesized, and is commercially available.
• The protein carbonyl groups are quantified by the new assay via a (stoichiometrically equimolar to the protein carbonyl hydrazone) standard curve, using the RBH reagent. The FTC assay, in contrast, although it claims a standard curve with FTC having equimolar stoichiometry with the protein carbonyl-FTC adduct. However, such a claim is unfounded, as no such data are provided by the referred study by the FTC-assay.

These findings are described in the articles entitled Protein carbonyl determination by a rhodamine B hydrazide-based fluorometric assay, and Protein and cell wall polysaccharide carbonyl determination by a neutral pH 2,4-dinitrophenylhydrazine-based photometric assay, recently published in the journal Redox Biology. This work was conducted by Christos D. Georgiou, Dimitrios Zisimopoulos, Vasiliki Argyropoulou, Electra Kalaitzopoulou, and Panayiotis V. Ioannou from the University of Patras, George Salachas from TEI of Western Greece, and Tilman Grune from the German Institute of Human Nutrition.