scholarly journals Real Time Normalization of Fast Photochemical Oxidation of Proteins Experiments by Inline Adenine Radical Dosimetry

2018 ◽  
Author(s):  
Joshua S. Sharp ◽  
Sandeep K. Misra ◽  
Jeffrey J. Persoff ◽  
Robert W. Egan ◽  
Scot R. Weinberger
Keyword(s):  
Hydroxyl Radical ◽  
Real Time ◽  
Hydroxyl Radicals ◽  
Conformational Changes ◽  
Radical Scavenging ◽  
Photochemical Oxidation ◽  
Major Step ◽  
Simple Method ◽  
Radical Concentration ◽  
High Concentrations

AbstractHydroxyl radical protein footprinting (HRPF) is a powerful method for measuring protein topography, allowing researchers to monitor events that alter the solvent accessible surface of a protein (e.g. ligand binding, aggregation, conformational changes, etc.) by measuring changes in the apparent rate of reaction of portions of the protein to hydroxyl radicals diffusing in solution. Fast Photochemical Oxidation of Proteins (FPOP) offers an ultra-fast benchtop method for performing HRPF, photolyzing hydrogen peroxide using a UV laser to generate high concentrations of hydroxyl radicals that are consumed on roughly a microsecond timescale. The broad reactivity of hydroxyl radicals means that almost anything added to the solution (e.g. ligands, buffers, excipients, etc.) will scavenge hydroxyl radicals, altering their half-life and changing the effective radical concentration experienced by the protein. Similarly, minute changes in peroxide concentration, laser fluence, and buffer composition can alter the effective radical concentration, making reproduction of data challenging. Here, we present a simple method for radical dosimetry that can be carried out as part of the FPOP workflow, allowing for measurement of effective radical concentration in real time. Additionally, by modulating the amount of radical generated, we demonstrate that FPOP HRPF experiments carried out in buffers with widely differing levels of hydroxyl radical scavenging capacity can be normalized on the fly, yielding statistically indistinguishable results for the same conformer. This method represents a major step in transforming FPOP into a robust and reproducible technology capable of probing protein structure in a wide variety of contexts.

scholarly journals Intrinsic Buffer Hydroxyl Radical Dosimetry Using Tris(Hydroxymethyl)Aminomethane

2019 ◽  
Author(s):  
Addison E. Roush ◽  
Mohammad Riaz ◽  
Sandeep K. Misra ◽  
Scot R. Weinberger ◽  
Joshua S. Sharp
Keyword(s):  
Hydroxyl Radical ◽  
Real Time ◽  
Hydroxyl Radicals ◽  
Flash Photolysis ◽  
Laser Flash Photolysis ◽  
Laser Flash ◽  
Photochemical Oxidation ◽  
Protein Surfaces ◽  
Radical Production ◽  
Experimental Parameters

AbstractFast Photochemical Oxidation of Proteins (FPOP) is a powerful covalent labeling tool that uses hydroxyl radicals generated by laser flash photolysis of hydrogen peroxide to footprint protein surfaces. Because radical production varies with many experimental parameters, hydroxyl radical dosimeters have been introduced to track the effective radical dosage experienced by the protein analyte. FPOP experiments performed using adenine optical radical dosimetry containing protein in Tris buffer demonstrated unusual dosimetry behavior. We have investigated the behavior of Tris under oxidative conditions in detail. We find that Tris can act as a novel gain-of-signal optical hydroxyl radical dosimeter in FPOP experiments. This new dosimeter is also amenable to inline real-time monitoring thereby allowing real-time adjustments to compensate for differences in samples for their quenching ability.

scholarly journals Towards reliable quantification of hydroxyl radicals in the Fenton reaction using chemical probes

RSC Advances ◽  
2018 ◽  
Vol 8 (10) ◽  
pp. 5321-5330 ◽  
Author(s):  
Burgos Castillo Rutely C. ◽  
Fontmorin Jean-M. ◽  
Tang Walter Z. ◽  
Dominguez-Benetton Xochitl ◽  
Sillanpää Mika
Keyword(s):  
Hydroxyl Radical ◽  
Hydroxyl Radicals ◽  
Fenton Reaction ◽  
Chemical Probes ◽  
Radical Concentration ◽  
Reliable Quantification

Quantification of hydroxyl radical concentration using two chemical probes was assessed through the Fenton reaction.

scholarly journals Hydroxyl Radical and Its Scavengers in Health and Disease

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Boguslaw Lipinski
Keyword(s):  
Oxidative Stress ◽  
Hydroxyl Radical ◽  
Hydroxyl Radicals ◽  
Disulfide Bonds ◽  
Radical Scavenging ◽  
Beneficial Effects ◽  
Binding Agents ◽  
Spatial Configurations ◽  
Health And Disease ◽  
Reducing Substances

It is generally believed that diseases caused by oxidative stress should be treated with antioxidants. However, clinical trials with such antioxidants as ascorbic acid and vitamin E, failed to produce the expected beneficial results. On the other hand, important biomolecules can be modified by the introduction of oxygen atoms by means of non-oxidative hydroxyl radicals. In addition, hydroxyl radicals can reduce disulfide bonds in proteins, specifically fibrinogen, resulting in their unfolding and scrambled refolding into abnormal spatial configurations. Consequences of this reaction are observed in many diseases such as atherosclerosis, cancer and neurological disorders, and can be prevented by the action of non-reducing substances. Moreover, many therapeutic substances, traditionally classified as antioxidants, accept electrons and thus are effective oxidants. It is described in this paper that hydroxyl radicals can be generated by ferric ions without any oxidizing agent. In view of the well-known damaging effect of poorly chelated iron in the human body, numerous natural products containing iron binding agents can be essential in the maintenance of human health. However, beneficial effects of the great number of phytochemicals that are endowed with hydroxyl radical scavenging and/or iron chelating activities should not be considered as a proof for oxidative stress.

Photochemical oxidation of municipal secondary effluents at low H2O2 dosage: Study of hydroxyl radical scavenging and process performance

2014 ◽  
Vol 237 ◽  
pp. 268-276 ◽  
Author(s):  
Bruno S. Souza ◽  
Renato F. Dantas ◽  
Angel Cruz ◽  
Carme Sans ◽  
Santiago Esplugas ◽  
...  
Keyword(s):  
Hydroxyl Radical ◽  
Radical Scavenging ◽  
Photochemical Oxidation ◽  
Process Performance ◽  
Secondary Effluents ◽  
Dosage Study

scholarly journals Radical Scavenging Activities of Novel Cationic Inulin Derivatives

Polymers ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 1295
Author(s):  
Yuan Chen ◽  
Yingqi Mi ◽  
Jingjing Zhang ◽  
Fang Dong ◽  
Qing Li ◽  
...  
Keyword(s):  
Hydroxyl Radical ◽  
Hydroxyl Radicals ◽  
Radical Scavenging Activity ◽  
Radical Scavenging ◽  
Synthesis Process ◽  
Scavenging Activity ◽  
Scavenging Effect ◽  
Radical Scavenging Effect ◽  
Ft Ir

Many saccharides are attractive targets for biomaterial applications, due to their abundance, biocompatibility, and biodegradability. In this article, a synthesis process of 6-N-substituted cationic inulin derivatives, including 6-pyridyl-6-deoxyinulin bromide (PIL), 6-(2-amino-pyridyl)-6-deoxyinulin bromide (2APIL), 6-(3-amino-pyridyl)-6-deoxyinulin bromide (3APIL), 6-(4-amino-pyridyl)-6-deoxyinulin bromide (4APIL), 6-(2,3-diamino-pyridyl)-6-deoxyinulin bromide (2,3DAPIL), 6-(3,4-diamino-pyridyl)-6-deoxyinulin bromide (3,4DAPIL), and 6-(2,6-diamino-pyridyl)-6-deoxyinulin bromide (2,6DAPIL) was described. The C6-OH of inulin was first activated by PPh3/N-bromosuccinimide (NBS) bromination. Then, pyridine and different kinds of amino-pyridine groups (different position and different numbers of amino) were grafted onto inulin, respectively, via nucleophilic substitution. Then, we confirmed their structure by Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy. After this, their radical scavenging activities against hydroxyl radical and diphenylpicryl phenylhydrazine (DPPH) radical were tested in vitro. Each derivative showed a distinct improvement in radical scavenging activity when compared to inulin. The hydroxyl-radical scavenging effect decreased in the following order: 3APIL > PIL > 3,4DAPIL > 4APIL > 2,3DAPIL > 2,6DAPIL > 2APIL. Amongst them, 3APIL revealed the most powerful scavenging effect on hydroxyl radicals, as well as DPPH radicals. At 1.6 mg/mL, it could completely eliminate hydroxyl radicals and could clear 65% of DPPH radicals. The results also showed that the steric hindrance effect and the substitute position of the amino group had an effect on the radical scavenging activity. Moreover, the application prospects of inulin derivatives as natural antioxidant biomaterials are scientifically proven in this paper.

scholarly journals Inhibition of microsomal oxidation of alcohols and of hydroxyl-radical-scavenging agents by the iron-chelating agent desferrioxamine

1983 ◽  
Vol 210 (1) ◽  
pp. 107-113 ◽  
Author(s):  
A I Cederbaum ◽  
E Dicker
Keyword(s):  
Hydroxyl Radical ◽  
Hydroxyl Radicals ◽  
Radical Scavenging ◽  
Chelating Agent ◽  
Dimethyl Sulphoxide ◽  
The Other ◽  
Radical Scavengers ◽  
Primary Alcohols ◽  
Hydroxyl Radical Scavengers ◽  
Oxidation Of Ethanol

Rat liver microsomes (microsomal fractions) catalyse the oxidation of straight-chain aliphatic alcohols and of hydroxyl-radical-scavenging agents during NADPH-dependent electron transfer. The iron-chelating agent desferrioxamine, which blocks the generation of hydroxyl radicals in other systems, was found to inhibit the following microsomal reactions: production of formaldehyde from either dimethyl sulphoxide or 2-methylpropan-2-ol (t-butylalcohol); generation of ethylene from 4-oxothiomethylbutyric acid; release of 14CO2 from [I-14C]benzoate; production of acetaldehyde from ethanol or butanal (butyraldehyde) from butan-1-ol. Desferrioxamine also blocked the increase in the oxidation of all these substrates produced by the addition of iron-EDTA to the microsomes. Desferrioxamine had no effect on a typical mixed-function-oxidase activity, the N-demethylation of aminopyrine, nor on the peroxidatic activity of catalase/H2O2 with ethanol. H2O2 appears to be the precursor of the oxidizing radical responsible for the oxidation of the alcohols and the other hydroxyl-radical scavengers. Chelation of microsomal iron by desferrioxamine most likely decreases the generation of hydroxyl radicals, which results in an inhibition of the oxidation of the alcohols and the hydroxyl-radical scavengers. Whereas desferrioxamine inhibited the oxidation of 2-methylpropan-2-ol, dimethyl sulphoxide, 4-oxothiomethylbutyrate and benzoate by more than 90%, the oxidation of ethanol and butanol could not be decreased by more than 45-60%. Higher concentrations of desferrioxamine were required to block the metabolism of the primary alcohols than to inhibit the metabolism of the other substrates. The desferrioxamine-insensitive rate of oxidation of ethanol was not inhibited by competitive hydroxyl-radical scavengers. These results suggest that primary alcohols may be oxidized by two pathways in microsomes, one dependent on the interaction of the alcohols with hydroxyl radicals (desferrioxamine-sensitive), the other which appears to be independent of these radicals (desferrioxamine-insensitive).

A simple method for assaying extracellular hydroxyl radical activity and its application to natural and synthetic waters

2003 ◽  
Vol 60 (2) ◽  
pp. 203-213 ◽  
Author(s):  
L A Molot ◽  
S A Miller ◽  
P J Dillon ◽  
C G Trick
Keyword(s):  
Hydroxyl Radical ◽  
Natural Waters ◽  
Culture Media ◽  
Cyanobacterial Bloom ◽  
Order Rate Constant ◽  
Photochemical Oxidation ◽  
Algal Culture ◽  
Simple Method ◽  
Oxidation Rates ◽  
Lake Waters

An assay has been developed to measure extracellular hydroxyl radical (OH*) activity in algal culture media and natural waters over a 4- to 5-day period. The first-order rate constant, k, for loss of absorbance at 590 or 620 nm was determined for erioglaucine, which is sensitive to OH*, insensitive to superoxide and hydrogen peroxide, and stable in the dark and under artificial radiation (280–750 nm) and solar radiation in the absence of oxidants. Variation in irradiance was accounted for by normalizing k with k for a ferric iron reference solution with dye (k/kfe). Trends in k/kfe for streams and lakes were consistent with previous data on photochemical oxidation rates of dissolved organic matter. Values for k/kfe were similar in filtered surface waters of eutrophic Heart Lake and nearby mesotrophic Lake St. George under artificial radiation. Hence, extracellular OH* did not appear to be a direct cause of the onset of a nuisance cyanobacterial bloom in Heart Lake, nor did OH* appear related to the absence of a bloom in Lake St. George. k/kfe was two orders of magnitude higher in algal culture media supplied with 8.8 mM nitrate than in lake waters.

Changes in the Antioxidant Activity of Purple Sweet Potato Wine during Storage

2011 ◽  
Vol 396-398 ◽  
pp. 1462-1465
Author(s):  
Wei Na Fu ◽  
Tuo Ping Li ◽  
You Feng Jia ◽  
Zhong Sheng Zhao ◽  
Wei Hou ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Hydroxyl Radical ◽  
Sweet Potato ◽  
Antioxidant Activities ◽  
Radical Scavenging Activity ◽  
Radical Scavenging ◽  
Scavenging Activity ◽  
Hydroxyl Radical Scavenging Activity ◽  
Purple Sweet Potato ◽  
High Concentrations

Anthocyanins, which possess strong antioxidant activities, are abundant in purple sweet potato wine. In the present study, changes in the antioxidant activity of purple sweet potato wine during storage were investigated. The results showed that purple sweet potato wine had high 2,2-dipheny-l-picrylhydrazyl (DPPH) free-radical and superoxide-radical scavenging activities, which were stable during storage periods. Compared with these two scavenging activities, the hydroxyl-radical scavenging activity of purple sweet potato wine was concentration-dependent and only presented relatively obvious scavenging activity at high concentrations. This hydroxyl-radical scavenging activity was also stabile during storage periods. The results are valuable in purple sweet potato wine research and development.

scholarly journals Self-Organized Amphiphiles are Poor Hydroxyl Radical Scavengers in Fast Photochemical Oxidation of Proteins Experiments

2020 ◽  
Author(s):  
Zhi Cheng ◽  
Charles Mobley ◽  
Sandeep K. Misra ◽  
Joshua S. Sharp
Keyword(s):  
Critical Micelle Concentration ◽  
Hydroxyl Radical ◽  
Radical Scavenging Activity ◽  
Radical Scavenging ◽  
Photochemical Oxidation ◽  
Radical Scavenger ◽  
Cellular Membranes ◽  
Scavenging Effect ◽  
Significant Difference ◽  
Hydroxyl Radical Scavenger

ABSTRACTThe analysis of membrane protein topography using fast photochemical oxidation of protein (FPOP) has been reported in recent years, but still underrepresented in literature. Based on the hydroxyl radical reactivity of lipids and other amphiphiles, it is believed that the membrane environment acts as a hydroxyl radical scavenger decreasing effective hydroxyl radical doses and resulting in less observed oxidation of proteins. Here, we investigated the effect of bulk hydroxyl radical scavenging in FPOP using both isolated cellular membranes as well as detergent micelles. We found no significant change in radical scavenging activity upon the addition of disrupted cellular membranes with the membrane concentration in the range of 0-25600 cell/μL using an inline radical dosimeter. We confirmed the non-scavenging nature of the membrane with the FPOP results of a soluble model protein in the presence of cell membranes, which showed no significant difference in oxidation with or without membranes. The use of detergents revealed that, while soluble detergent below the critical micelle concentration acts as a potent hydroxyl radical scavenger as expected, additional detergent has little to no hydroxyl radical scavenging effect once the critical micelle concentration is reached. These results suggest that any scavenging effect of membranes or organized amphiphilic membrane mimetics in FPOP experiments are not due to bulk hydroxyl radical scavenging, but may be due to a localized scavenging phenomenon.

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