Comparison of the ribonucleolytic activity of the dityrosine cross-linked Ribonuclease A dimer with its monomer in the presence of inhibitors

Dinda, Amit Kumar; Tripathy, Debi Ranjan; Das, Anushree; Dasgupta, Swagata

doi:10.1016/j.ijbiomac.2013.10.033

Cited by 14 publications

(14 citation statements)

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“…A previous study on the enzymatic activity of the RNase A dimer showed that the photoirradiated DT cross-linked dimer has lower substrate affinity than the RNase A monomer. 53 …”

Section: Results and Discussionmentioning

confidence: 99%

“… 52 Dityrosine (DT) formation can result in intermolecular covalent bond formation between two Tyr residues of different monomers, leading to a protein dimer. 53 Because both fullerene and fullerenol have antioxidant properties, these molecules have been used to study the changes in DT formation caused due to oxidation of RNase A in the presence of peroxide. The extent of dimer formation has been monitored using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF).…”

Section: Introductionmentioning

confidence: 99%

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Exploring the Inhibitory and Antioxidant Effects of Fullerene and Fullerenol on Ribonuclease A

et al. 2018

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Fullerene–protein interaction studies have been a key topic of investigation in recent times, but the lower water solubility of fullerene somewhat limits its application in the biological system. In this work, we have compared the activities of fullerene and its water-soluble hydrated form, that is fullerenol, on ribonuclease A (RNase A) under physiological conditions (pH 7.4). The interaction studies of fullerene and fullerenol with protein suggest that the binding depends on the hydrophobic interactions between the protein and the ligand. In addition, fullerene and fullerenol slow down the ribonucleolytic activity of RNase A through noncompetitive and mixed types of inhibition, respectively. This precisely gives the idea about the ligand-binding sites in RNase A, which has further been explored using docking studies. Both these nanoparticles show a reduction in dityrosine formation in RNase A caused due to oxidative stress and also prevent RNase A dimer formation to different extents depending on their concentration.

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“…A previous study on the enzymatic activity of the RNase A dimer showed that the photoirradiated DT cross-linked dimer has lower substrate affinity than the RNase A monomer. 53 …”

Section: Results and Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Exploring the Inhibitory and Antioxidant Effects of Fullerene and Fullerenol on Ribonuclease A

et al. 2018

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“…Nitration of protein residues often results in the loss of enzymatic activity and in order to study this, we have carried out the enzyme kinetics study of nitrated RNase A (RNase A NT ) under different conditions and compared the results with that of native RNase A. In each of the cases we have monitored the values of maximum velocity ( V max ) and Michaelis–Menten constant ( K M ) which determines the rate of enzyme substrate reaction and the affinity towards the substrate, respectively . It can be observed that the maximum velocity reduces by ≈11 % when RNase A is treated with PN at pH 6.0 [RNase A NT (pH 6.0)] whereas at pH 7.4 [RNase A NT (pH 7.4)] it reduces by ≈23 % [Table ].…”

Section: Resultsmentioning

confidence: 99%

“…In each of the cases we have monitored the values of maximum velocity( V max )a nd Michaelis-Menten constant (K M )w hich determines the rate of enzymes ubstrate reaction and the affinity towards the substrate, respectively. [62,64] It can be observed that the maximum velocityr educes by % 11 % when RNase Ai st reated with PN at pH 6.0 [RNase A NT (pH 6.0)] whereas at pH 7.4 [RNase A NT (pH 7.4)] it reduces by % 23 % [ Table 3]. O nt he other hand, the higherv alue of K M for pH 6.0 and 7.4 indicates that the affinity of the enzyme towards the substrate (2',3'-cCMP) reduces upon nitration.…”

Section: Enzyme Inhibition Kineticsmentioning

confidence: 99%

pH‐Dependent Nitrotyrosine Formation in Ribonuclease A is Enhanced in the Presence of Polyethylene Glycol (PEG)

Roy

Panda

Hati

et al. 2019

Chemistry An Asian Journal

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Protein nitration can occur as ar esult of peroxynitrite-mediated oxidative stress. Excess production of peroxynitrite (PN) within the cellular mediumc an cause oxidative damage to biomolecules.T he in vitro nitration of Ribonuclease A( RNase A) resultsi nn itrotyrosine (NT) formation with as trong dependence on the pH of the medium. In order to mimic the cellular environment in this study,P Nmediated RNase An itration has been carried out in ac rowd-ed medium. The degree of nitration is higher at pH 7.4 (physiological pH) compared to pH 6.0 (tumorc ell pH). The extent of nitration increases significantly when PN is added to RNase Ai nt he presence of crowding agentsP EG 400 and PEG 6000. PEG has been found to stabilize PN overaprolonged period,t hereby increasing the degree of nitration. NT formation in RNase Aa lso results in as ignificant loss in enzymatic activity.Scheme1.Generation of peroxynitrite and subsequent formation of nitrating species.

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“…This laboratory has reported the decrease in enzymatic activity of photoirradiated RNase A in comparison to the monomer. [23] The lowering of activity is mainly due to the formation of the RNase A dimer caused by dityrosine bond formation which gives an intense fluorescence peak around 405 nm when excited at 317 nm.…”

Section: Introductionmentioning

confidence: 99%

β‐cyclodextrin encapsulated polyphenols as effective antioxidants

et al. 2017

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Formation of dityrosine (DT) cross-linkages in proteins is one of the most widely used markers of oxidative stress. Ribonuclease A (RNase A) has 6 Tyr residues and shows a characteristic DT fluorescence peak upon oxidation in addition to major changes in its secondary structure. DT formation can be prevented by using polyphenols (GA, ECG, and EGCG) which are known to have strong antioxidant activity. However, it has been observed that ECG and EGCG initiate protein oligomerization due to protein-polyphenol cross-linkages. To prevent the formation of such crosslinkages we have used b-cyclodextrin (b-CD) to encapsulate the polyphenols and studied its antioxidant properties along with that of free polyphenols. The polyphenol/b-cyclodextrin (b-CD) inclusion complexes not only prevent DT formation but also reduce protein oligomerization. This may be attributed to the fact that the quinone forming rings of ECG and EGCG become encapsulated in the cavity of b-CD and are no longer available for protein cross-linking.b-cyclodextrin encapsulation, dityrosine, oxidative stress, polyphenols, protein oligomerization | I N TR ODU C TI ONBiological aging in mammalian cells is a direct indication of oxidative stress in proteins. [1] These oxidatively modified forms of proteins are generated due to the presence of reactive oxygen species (ROS) such as hydroxyl and superoxide free radicals. [2,3] Persulfates, a key ingredient in flour, are a source of sulfate (SO 2 4 ) free radicals under neutral pH. [4,5] Due to the higher oxidizing power of the sulfate radical (E 0 5 12.60 V) and longer half-life period over the OH radical, it has drawn attention in recent times as a powerful oxidizing agent. [6][7][8] Many chemical and cosmetic industries require persulfates as their key components [9] and workers often suffer from skin and respiratory diseases due to hypersensitive reactions. [10] Generally, the lower thermal energy at room temperature (25-508C) is not sufficient enough to activate persulfate, but generation of SO 2 4 radical is facilitated in presence of metal cations. Since proteins tend to lose their structure and function at high temperatures, [11] we have carried out the oxidation of ribonuclease A (RNase A) in presence of persulfate using cobalt (II) ion at 378C. [12] Cobalt is an integral component of vitamin B12 and thus physiologically found in most tissues mainly in liver and kidney. [13,14] Gill et al., reported the formation of oligomeric species (dimer, trimer, tetramer) when RNase A was exposed to KHSO 5 in presence of metal ions such as Ni 21 and Cu 21 and the high molecular weight species were identified using SDS-PAGE. [12] An important biomarker of oxidative stress is the dityrosine moiety generated due to the covalent linkage of two Tyr residues. [15] Dityrosine bond formation is an indication of aging, and disease related problems since it generally occurs during amyloid fibril formation, [16] Parkinson disease, [17] cerebrospinal fluid damage, [18] and also in the cataract of the eye lens. [19,20] D...

show abstract

Comparison of the ribonucleolytic activity of the dityrosine cross-linked Ribonuclease A dimer with its monomer in the presence of inhibitors

Cited by 14 publications

References 48 publications

Exploring the Inhibitory and Antioxidant Effects of Fullerene and Fullerenol on Ribonuclease A

Exploring the Inhibitory and Antioxidant Effects of Fullerene and Fullerenol on Ribonuclease A

pH‐Dependent Nitrotyrosine Formation in Ribonuclease A is Enhanced in the Presence of Polyethylene Glycol (PEG)

β‐cyclodextrin encapsulated polyphenols as effective antioxidants

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