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Michael L. Gross - One of the best experts on this subject based on the ideXlab platform.
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protein higher order structure determination by fast Photochemical Oxidation of proteins and mass spectrometry analysis
Nature Protocols, 2020Co-Authors: Xiaoran Roger Liu, Don L Rempel, Michael L. GrossAbstract:The higher-order structure (HOS) of proteins plays a critical role in their function; therefore, it is important to our understanding of their function that we have as much information as possible about their three-dimensional structure and how it changes with time. Mass spectrometry (MS) has become an important tool for determining protein HOS owing to its high throughput, mid-to-high spatial resolution, low sample amount requirement and broad compatibility with various protein systems. Modern MS-based protein HOS analysis relies, in part, on footprinting, where a reagent reacts 'to mark' the solvent-accessible surface of the protein, and MS-enabled proteomic analysis locates the modifications to afford a footprint. Fast Photochemical Oxidation of proteins (FPOP), first introduced in 2005, has become a powerful approach for protein footprinting. Laser-induced hydrogen peroxide photolysis generates hydroxyl radicals that react with solvent-accessible side chains (14 out of 20 amino acid side chains) to fulfill the footprinting. The reaction takes place at sub-milliseconds, faster than most of labeling-induced protein conformational changes, thus enabling a 'snapshot' of protein HOS in solution. As a result, FPOP has been employed in solving several important problems, including mapping epitopes, following protein aggregation, locating small molecule binding, measuring ligand-binding affinity, monitoring protein folding and unfolding and determining hidden conformational changes invisible to other methods. Broader adoption will be promoted by dissemination of the technical details for assembling the FPOP platform and for dealing with the complexities of analyzing FPOP data. In this protocol, we describe the FPOP platform, the conditions for successful footprinting and its examination by mass measurements of the intact protein, the post-labeling sample handling and digestion, the liquid chromatography-tandem MS analysis of the digested sample and the data analysis with Protein Metrics Suite. This protocol is intended not only as a guide for investigators trying to establish an FPOP platform in their own lab but also for those willing to incorporate FPOP as an additional tool in addressing their questions of interest.
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hydroxyl radical reaction pathways for the fast Photochemical Oxidation of proteins platform as revealed by 18o isotopic labeling
Analytical Chemistry, 2019Co-Authors: Xiaoran Roger Liu, Mengru Mira Zhang, Bojie Zhang, Don L Rempel, Michael L. GrossAbstract:Fast Photochemical Oxidation of protein (FPOP) has become an important mass spectrometry-based protein footprinting approach. Although the hydroxyl radical (•OH) generated by photolysis of hydrogen peroxide (H2O2) is most commonly used, the pathways for its reaction with amino-acid side chains remain unclear. Here, we report a systematic study of •OH oxidative modification of 13 amino acid residues by using 18O isotopic labeling. The results differentiate three classes of residues on the basis of their oxygen uptake preference toward different oxygen sources. Histidine, arginine, tyrosine, and phenylalanine residues preferentially take oxygen from H2O2. Methionine residues competitively take oxygen from H2O2 and dissolved oxygen (O2), whereas the remaining residues take oxygen exclusively from O2. Results reported in this work deepen the understanding of •OH labeling pathway on a FPOP platform, opening new possibilities for tailoring FPOP conditions in addressing many biological questions in a profound way.
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Protein-Ligand Interaction by Ligand Titration, Fast Photochemical Oxidation of Proteins and Mass Spectrometry: LITPOMS
Journal of The American Society for Mass Spectrometry, 2019Co-Authors: Xiaoran Roger Liu, Mengru Mira Zhang, Don L Rempel, Michael L. GrossAbstract:We report a novel method named LITPOMS (ligand titration, fast Photochemical Oxidation of proteins and mass spectrometry) to characterize protein-ligand binding stoichiometry, binding sites, and site-specific binding constants. The system used to test the method is melittin–calmodulin, in which the peptide melittin binds to calcium-bound calmodulin. Global-level measurements reveal the binding stoichiometry of 1:1 whereas peptide-level data coupled with fitting reveal the binding sites and the site-specific binding affinity. Moreover, we extended the analysis to the residue level and identified six critical binding residues. The results show that melittin binds to the N-terminal, central linker, and C-terminal regions of holo-calmodulin with an affinity of 4.6 nM, in agreement with results of previous studies. LITPOMS, for the first time, brings high residue-level resolution to affinity measurements, providing simultaneously qualitative and quantitative understanding of protein-ligand binding. The approach can be expanded to other binding systems without tagging the protein to give high spatial resolution. Graphical Abstract
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a fast Photochemical Oxidation of proteins fpop platform for free radical reactions the carbonate radical anion with peptides and proteins
Free Radical Biology and Medicine, 2019Co-Authors: Mengru Mira Zhang, Don L Rempel, Michael L. GrossAbstract:Abstract Fast Photochemical Oxidation of Protein (FPOP), based on a pulsed KrF laser (248 nm) for free-radical generation, is a biophysical method that utilizes hydroxyl radicals to footprint proteins in solution. FPOP has been recognized for structural proteomics investigations, including epitope mapping, protein-aggregation characterization, protein-folding monitoring, and binding-affinity determination. The distinct merits of the platform are: i) the use of a scavenger to control radical lifetime and allow fast (“snapshot”) footprinting of solvent-accessible residues in a protein; ii) the employment of a flow system to enable single-shot irradiation of small plugs of the targeted sample; iii) the use of methionine and catalase after radical Oxidation chemistry to prevent post-Oxidation with residual oxidizing species; and iv) the utilization of mature mass spectrometry-based proteomic methods to afford detailed analysis. In addition to •OH, other reactive reagents (e.g., carbenes, iodide, sulfate radical anion, and trifluoromethyl radical) can be implemented on this platform to increase the versatility and scope. In this study, we further elaborate the use of FPOP platform to generate secondary radicals and establish a workflow to answer fundamental questions regarding the intrinsic selectivity and reactivity of radicals that are important in biology. Carbonate radical anion is the example we chose owing to its oxidative character and important putative pathogenic roles in inflammation. This systematic study with model proteins/peptides gives consistent results with a previous study that evaluated reactivity with free amino acids and shows that methionine and tryptophan are the most reactive residues with CO3-•. Other aromatic amino acids (i.e., tyrosine, histidine and phenylalanine) exhibit moderate reactivity, whereas, aliphatic amino acids are inert, unlike with •OH. The outcome demonstrates this approach to be appropriate for studying the fast reactions of radicals with proteins.
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recognition of human igg1 by fcγ receptors structural insights from hydrogen deuterium exchange and fast Photochemical Oxidation of proteins coupled with mass spectrometry
Biochemistry, 2019Co-Authors: Liuqing Shi, Michael L. Gross, Tun Liu, Yining HuangAbstract:Antibody-dependent cell-mediated cytotoxicity (ADCC) is an effector function of immunoglobulins (IgGs) involved in the killing of target cells by a cytotoxic effector cell. Recognition of IgG by Fc receptors expressed on natural killer cells, mostly FcγIII receptors (FcγRIII), underpins the ADCC mechanism, thus motivating investigations of these interactions. In this paper, we describe the combination of hydrogen-deuterium exchange and fast Photochemical Oxidation of proteins (FPOP) coupled with mass spectrometry to study the interactions of the human IgG1/FcγRIII complex. Using these orthogonal approaches, we identified critical peptide regions and residues involved in the recognition of IgG1 by FcγRIII. The footprinting results are consistent with the previously published crystal structure of the IgG1 Fc/FcγRIII complex. Additionally, our FPOP results reveal the conformational changes in the Fab domain upon binding of the Fc domain to FcγRIII. These data demonstrate the value of footprinting as part of a comprehensive toolbox for identifying the changes in the higher-order structure of therapeutic antibodies in solution.
Don L Rempel - One of the best experts on this subject based on the ideXlab platform.
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protein higher order structure determination by fast Photochemical Oxidation of proteins and mass spectrometry analysis
Nature Protocols, 2020Co-Authors: Xiaoran Roger Liu, Don L Rempel, Michael L. GrossAbstract:The higher-order structure (HOS) of proteins plays a critical role in their function; therefore, it is important to our understanding of their function that we have as much information as possible about their three-dimensional structure and how it changes with time. Mass spectrometry (MS) has become an important tool for determining protein HOS owing to its high throughput, mid-to-high spatial resolution, low sample amount requirement and broad compatibility with various protein systems. Modern MS-based protein HOS analysis relies, in part, on footprinting, where a reagent reacts 'to mark' the solvent-accessible surface of the protein, and MS-enabled proteomic analysis locates the modifications to afford a footprint. Fast Photochemical Oxidation of proteins (FPOP), first introduced in 2005, has become a powerful approach for protein footprinting. Laser-induced hydrogen peroxide photolysis generates hydroxyl radicals that react with solvent-accessible side chains (14 out of 20 amino acid side chains) to fulfill the footprinting. The reaction takes place at sub-milliseconds, faster than most of labeling-induced protein conformational changes, thus enabling a 'snapshot' of protein HOS in solution. As a result, FPOP has been employed in solving several important problems, including mapping epitopes, following protein aggregation, locating small molecule binding, measuring ligand-binding affinity, monitoring protein folding and unfolding and determining hidden conformational changes invisible to other methods. Broader adoption will be promoted by dissemination of the technical details for assembling the FPOP platform and for dealing with the complexities of analyzing FPOP data. In this protocol, we describe the FPOP platform, the conditions for successful footprinting and its examination by mass measurements of the intact protein, the post-labeling sample handling and digestion, the liquid chromatography-tandem MS analysis of the digested sample and the data analysis with Protein Metrics Suite. This protocol is intended not only as a guide for investigators trying to establish an FPOP platform in their own lab but also for those willing to incorporate FPOP as an additional tool in addressing their questions of interest.
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hydroxyl radical reaction pathways for the fast Photochemical Oxidation of proteins platform as revealed by 18o isotopic labeling
Analytical Chemistry, 2019Co-Authors: Xiaoran Roger Liu, Mengru Mira Zhang, Bojie Zhang, Don L Rempel, Michael L. GrossAbstract:Fast Photochemical Oxidation of protein (FPOP) has become an important mass spectrometry-based protein footprinting approach. Although the hydroxyl radical (•OH) generated by photolysis of hydrogen peroxide (H2O2) is most commonly used, the pathways for its reaction with amino-acid side chains remain unclear. Here, we report a systematic study of •OH oxidative modification of 13 amino acid residues by using 18O isotopic labeling. The results differentiate three classes of residues on the basis of their oxygen uptake preference toward different oxygen sources. Histidine, arginine, tyrosine, and phenylalanine residues preferentially take oxygen from H2O2. Methionine residues competitively take oxygen from H2O2 and dissolved oxygen (O2), whereas the remaining residues take oxygen exclusively from O2. Results reported in this work deepen the understanding of •OH labeling pathway on a FPOP platform, opening new possibilities for tailoring FPOP conditions in addressing many biological questions in a profound way.
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Protein-Ligand Interaction by Ligand Titration, Fast Photochemical Oxidation of Proteins and Mass Spectrometry: LITPOMS
Journal of The American Society for Mass Spectrometry, 2019Co-Authors: Xiaoran Roger Liu, Mengru Mira Zhang, Don L Rempel, Michael L. GrossAbstract:We report a novel method named LITPOMS (ligand titration, fast Photochemical Oxidation of proteins and mass spectrometry) to characterize protein-ligand binding stoichiometry, binding sites, and site-specific binding constants. The system used to test the method is melittin–calmodulin, in which the peptide melittin binds to calcium-bound calmodulin. Global-level measurements reveal the binding stoichiometry of 1:1 whereas peptide-level data coupled with fitting reveal the binding sites and the site-specific binding affinity. Moreover, we extended the analysis to the residue level and identified six critical binding residues. The results show that melittin binds to the N-terminal, central linker, and C-terminal regions of holo-calmodulin with an affinity of 4.6 nM, in agreement with results of previous studies. LITPOMS, for the first time, brings high residue-level resolution to affinity measurements, providing simultaneously qualitative and quantitative understanding of protein-ligand binding. The approach can be expanded to other binding systems without tagging the protein to give high spatial resolution. Graphical Abstract
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a fast Photochemical Oxidation of proteins fpop platform for free radical reactions the carbonate radical anion with peptides and proteins
Free Radical Biology and Medicine, 2019Co-Authors: Mengru Mira Zhang, Don L Rempel, Michael L. GrossAbstract:Abstract Fast Photochemical Oxidation of Protein (FPOP), based on a pulsed KrF laser (248 nm) for free-radical generation, is a biophysical method that utilizes hydroxyl radicals to footprint proteins in solution. FPOP has been recognized for structural proteomics investigations, including epitope mapping, protein-aggregation characterization, protein-folding monitoring, and binding-affinity determination. The distinct merits of the platform are: i) the use of a scavenger to control radical lifetime and allow fast (“snapshot”) footprinting of solvent-accessible residues in a protein; ii) the employment of a flow system to enable single-shot irradiation of small plugs of the targeted sample; iii) the use of methionine and catalase after radical Oxidation chemistry to prevent post-Oxidation with residual oxidizing species; and iv) the utilization of mature mass spectrometry-based proteomic methods to afford detailed analysis. In addition to •OH, other reactive reagents (e.g., carbenes, iodide, sulfate radical anion, and trifluoromethyl radical) can be implemented on this platform to increase the versatility and scope. In this study, we further elaborate the use of FPOP platform to generate secondary radicals and establish a workflow to answer fundamental questions regarding the intrinsic selectivity and reactivity of radicals that are important in biology. Carbonate radical anion is the example we chose owing to its oxidative character and important putative pathogenic roles in inflammation. This systematic study with model proteins/peptides gives consistent results with a previous study that evaluated reactivity with free amino acids and shows that methionine and tryptophan are the most reactive residues with CO3-•. Other aromatic amino acids (i.e., tyrosine, histidine and phenylalanine) exhibit moderate reactivity, whereas, aliphatic amino acids are inert, unlike with •OH. The outcome demonstrates this approach to be appropriate for studying the fast reactions of radicals with proteins.
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implementing fast Photochemical Oxidation of proteins fpop as a footprinting approach to solve diverse problems in structural biology
Methods, 2018Co-Authors: Bojie Zhang, Don L Rempel, Ming Cheng, Michael L. GrossAbstract:Fast Photochemical Oxidation of proteins (FPOP) is a footprinting technique used in mass spectrometry-based structural proteomics. It has been applied to solve a variety of problems in different areas of biology. A FPOP platform requires a laser, optics, and sample flow path properly assembled to enable fast footprinting. Sample preparation, buffer conditions, and reagent concentrations are essential to obtain reasonable Oxidations on proteins. FPOP samples can be analyzed by LC-MS methods to measure the modification extent, which is a function of the solvent-accessible surface area of the protein. The platform can be expanded to accommodate several new approaches, including dose-response studies, new footprinting reagents, and two-laser pump-probe experiments. Here, we briefly review FPOP applications and in a detailed manner describe the procedures to set up an FPOP protein footprinting platform.
Brian Gau - One of the best experts on this subject based on the ideXlab platform.
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fast Photochemical Oxidation of proteins and mass spectrometry follow submillisecond protein folding at the amino acid level
Journal of the American Chemical Society, 2012Co-Authors: Jiawei Chen, Don L Rempel, Brian Gau, Michael L. GrossAbstract:We report a study of submillisecond protein folding with amino-acid residue resolution achieved with a two-laser pump/probe experiment with analysis by mass spectrometry. The folding of a test protein, barstar, can be triggered by a laser-induced temperature jump (T jump) from ∼0 °C to ∼room temperature. Subsequent reactions via fast Photochemical Oxidation of proteins (FPOP) at various fractional millisecond points after the T jump lead to oxidative modification of solvent-accessible side chains whose “protection” changes with time and extent of folding. The modifications are identified and quantified by LC-MS/MS following proteolysis. Among all the segments that form secondary structure in the native state, helix1 shows a decreasing trend of oxidative modification during the first 0.1–1 ms of folding while others do not change in this time range. Residues I5, H17, L20, L24 and F74 are modified less in the intermediate state than the denatured state, likely due to full or partial protection of these resi...
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fast Photochemical Oxidation of proteins and mass spectrometry follow submillisecond protein folding at the amino acid level
Journal of the American Chemical Society, 2012Co-Authors: Jiawei Chen, Don L Rempel, Brian Gau, Michael L. GrossAbstract:We report a study of submillisecond protein folding with amino-acid residue resolution achieved with a two-laser pump/probe experiment with analysis by mass spectrometry. The folding of a test protein, barstar, can be triggered by a laser-induced temperature jump (T jump) from ∼0 °C to ∼room temperature. Subsequent reactions via fast Photochemical Oxidation of proteins (FPOP) at various fractional millisecond points after the T jump lead to oxidative modification of solvent-accessible side chains whose "protection" changes with time and extent of folding. The modifications are identified and quantified by LC-MS/MS following proteolysis. Among all the segments that form secondary structure in the native state, helix(1) shows a decreasing trend of oxidative modification during the first 0.1-1 ms of folding while others do not change in this time range. Residues I5, H17, L20, L24 and F74 are modified less in the intermediate state than the denatured state, likely due to full or partial protection of these residues as folding occurs. We propose that in the early folding stage, barstar forms a partially solvent-accessible hydrophobic core consisting of several residues that have long-range interaction with other, more remote residues in the protein sequence. Our data not only are consistent with the previous conclusion that barstar fast folding follows the nucleation-condensation mechanism with the nucleus centered on helix(1) formed in a folding intermediate but also show the efficacy of this new approach to following protein folding on the submillisecond time range.
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fast Photochemical Oxidation of proteins for comparing structures of protein ligand complexes the calmodulin peptide model system
Analytical Chemistry, 2011Co-Authors: Hao Zhang, Lisa M Jones, Brian Gau, Ilan Vidavsky, Michael L. GrossAbstract:Fast Photochemical Oxidation of proteins (FPOP) is a mass spectrometry-based protein footprinting method that modifies proteins on the microsecond time scale. Highly reactive •OH, produced by laser photolysis of hydrogen peroxide, oxidatively modifies the side chains of approximately one-half the common amino acids on this time scale. Because of the short labeling exposure, only solvent-accessible residues are sampled. Quantification of the modification extent for the apo and holo states of a protein−ligand complex provides structurally sensitive information at the amino-acid level to compare the structures of unknown protein complexes with known ones. We report here the use of FPOP to monitor the structural changes of calmodulin in its established binding to M13 of the skeletal muscle myosin light chain kinase. We use the outcome to establish the unknown structures resulting from binding with melittin and mastoparan. The structural comparison follows a comprehensive examination of the extent of FPOP modi...
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sulfate radical anion as a new reagent for fast Photochemical Oxidation of proteins
Analytical Chemistry, 2010Co-Authors: Brian Gau, Hao Chen, Yun Zhang, Michael L. GrossAbstract:The focus is to expand the original design of fast Photochemical Oxidation of proteins (FPOP) and introduce SO4−•, generated by 248 nm homolysis of low millimolar levels of persulfate, as a radical reactant in protein footprinting. FPOP is a chemical approach to footprinting proteins and protein complexes by “snapshot” reaction with free radicals. The radical used until now is the OH radical, and it provides a measure of residue-resolved solvent accessibility of the native protein. We show that FPOP can accommodate other reagents, increasing its versatility. The new persulfate FPOP system is a potent, nonspecific, and tunable footprinting method; 3−5 times less persulfate is needed to give the same global levels of modification as seen with OH radicals. Although solvent-exposed His and Tyr residues are more reactive with SO4−• than with •OH, Oxidation of apomyoglobin and calmodulin shows that •OH probes smaller accessible areas than SO4−•, with the possible exception of histidine. His64, an axial ligand i...
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fast Photochemical Oxidation of protein footprints faster than protein unfolding
Analytical Chemistry, 2009Co-Authors: Brian Gau, Don L Rempel, Joshua S Sharp, Michael L. GrossAbstract:Fast Photochemical Oxidation of proteins (FPOP) is a chemical footprinting method whereby exposed amino-acid residues are covalently labeled by Oxidation with hydroxyl radicals produced by the photolysis of hydrogen peroxide. Modified residues can be detected by standard trypsin proteolysis followed by LC/MS/MS, providing information about solvent accessibility at the peptide and even the amino-acid level. Like other chemical footprinting techniques, FPOP must ensure only the native conformation is labeled. Although Oxidation via hydroxyl radical induces unfolding in proteins on a time scale of milliseconds or longer, FPOP is designed to limit •OH exposure to 1 μs or less by employing a pulsed laser for initiation to produce the radicals and a radical-scavenger to limit their lifetimes. We applied FPOP to three Oxidation-sensitive proteins and found that the distribution of modification (Oxidation) states is Poisson when a scavenger is present, consistent with a single conformation protein modification mo...
Jiawei Chen - One of the best experts on this subject based on the ideXlab platform.
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fast Photochemical Oxidation of proteins and mass spectrometry follow submillisecond protein folding at the amino acid level
Journal of the American Chemical Society, 2012Co-Authors: Jiawei Chen, Don L Rempel, Brian Gau, Michael L. GrossAbstract:We report a study of submillisecond protein folding with amino-acid residue resolution achieved with a two-laser pump/probe experiment with analysis by mass spectrometry. The folding of a test protein, barstar, can be triggered by a laser-induced temperature jump (T jump) from ∼0 °C to ∼room temperature. Subsequent reactions via fast Photochemical Oxidation of proteins (FPOP) at various fractional millisecond points after the T jump lead to oxidative modification of solvent-accessible side chains whose “protection” changes with time and extent of folding. The modifications are identified and quantified by LC-MS/MS following proteolysis. Among all the segments that form secondary structure in the native state, helix1 shows a decreasing trend of oxidative modification during the first 0.1–1 ms of folding while others do not change in this time range. Residues I5, H17, L20, L24 and F74 are modified less in the intermediate state than the denatured state, likely due to full or partial protection of these resi...
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fast Photochemical Oxidation of proteins and mass spectrometry follow submillisecond protein folding at the amino acid level
Journal of the American Chemical Society, 2012Co-Authors: Jiawei Chen, Don L Rempel, Brian Gau, Michael L. GrossAbstract:We report a study of submillisecond protein folding with amino-acid residue resolution achieved with a two-laser pump/probe experiment with analysis by mass spectrometry. The folding of a test protein, barstar, can be triggered by a laser-induced temperature jump (T jump) from ∼0 °C to ∼room temperature. Subsequent reactions via fast Photochemical Oxidation of proteins (FPOP) at various fractional millisecond points after the T jump lead to oxidative modification of solvent-accessible side chains whose "protection" changes with time and extent of folding. The modifications are identified and quantified by LC-MS/MS following proteolysis. Among all the segments that form secondary structure in the native state, helix(1) shows a decreasing trend of oxidative modification during the first 0.1-1 ms of folding while others do not change in this time range. Residues I5, H17, L20, L24 and F74 are modified less in the intermediate state than the denatured state, likely due to full or partial protection of these residues as folding occurs. We propose that in the early folding stage, barstar forms a partially solvent-accessible hydrophobic core consisting of several residues that have long-range interaction with other, more remote residues in the protein sequence. Our data not only are consistent with the previous conclusion that barstar fast folding follows the nucleation-condensation mechanism with the nucleus centered on helix(1) formed in a folding intermediate but also show the efficacy of this new approach to following protein folding on the submillisecond time range.
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temperature jump and fast Photochemical Oxidation probe submillisecond protein folding
Journal of the American Chemical Society, 2010Co-Authors: Jiawei Chen, Don L Rempel, Michael L. GrossAbstract:We report a new mass-spectrometry-based approach for studying protein-folding dynamics on the submillisecond time scale. The strategy couples a temperature jump with fast Photochemical Oxidation of proteins (FPOP), whereby folding/unfolding is followed by changes in oxidative modifications by OH radical reactions. Using a flow system containing the protein barstar as a model, we altered the protein’s equilibrium conformation by applying the temperature jump and demonstrated that its reactivity with OH free radicals serves as a reporter of the conformational change. Furthermore, we found that the time-dependent increase in mass resulting from free-radical Oxidation is a measure of the rate constant for the transition from the unfolded to the first intermediate state. This advance offers the promise that, when extended with mass-spectrometry-based proteomic analysis, the sites and kinetics of folding/unfolding can also be followed on the submillisecond time scale.
Patrick Da Costa - One of the best experts on this subject based on the ideXlab platform.
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tio2 clay as a heterogeneous catalyst in photocatalytic Photochemical Oxidation of anionic reactive blue 19
Arabian Journal of Chemistry, 2014Co-Authors: Haithem Bel Hadjltaief, Maria Elena Galvez, Mourad Ben Zina, Patrick Da CostaAbstract:Abstract A TiO2-coated Tunisian clay (TiO2–clay) was synthesized by a typical impregnation method. The physicochemical characterization points to a successful impregnation of titania on the clay surface. The activity of this structured catalyst was studied in the photocatalytic/Photochemical Oxidation of anionic reactive blue 19 (RB 19). The effect of UVA and solar irradiation (UV-solar) was studied at room temperature. TiO2–clay demonstrated an effective degradation of RB 19 under both types of irradiation. Moreover, in this study, the effects of various oxidants such as hydrogen peroxide (H2O2), potassium peroxodisulfate (K2S2O8) and sodium carbonate (Na2CO3) were thoroughly investigated. H2O2 was a promising oxidant for promoting RB 19 degradation under UVA. The kinetics of discoloration of RB 19 followed a pseudo-first-order rate law. We can remark that 20 min of UV irradiation was enough to achieve 100% discoloration of the aqueous solution. However, under UV–Vis, HPLC and chemical oxygen demand measurements indicated, that a longer reaction time (of around 45 min) was required for achieving the complete dye mineralization. The findings clearly demonstrated the applicability of this TiO2/clay catalyst for the photocatalytic Oxidation of RB 19.