Pengjun Zhang

The interaction between 4-aminoantipyrine and bovine serum albumin: Multiple spectroscopic and molecular docking investigations

4-Aminoantipyrine (AAP) is widely used in the pharmaceutical industry, in biochemical experiments and in environmental monitoring. AAP as an aromatic pollutant in the environment poses a great threat to human health. To evaluate the toxicity of AAP at the protein level, the effects of AAP on bovine serum albumin (BSA) were investigated by multiple spectroscopic techniques and molecular modeling. After the inner filter effect was eliminated, the experimental results showed that AAP effectively quenched the intrinsic fluorescence of BSA via static quenching. The number of binding sites, the binding constant, the thermodynamic parameters and binding subdomain were measured, and indicated that AAP could spontaneously bind with BSA on subdomain IIIA through electrostatic forces. Molecular docking results revealed that AAP interacted with the Glu 488 and Glu 502 residues of BSA. Furthermore, the conformation of BSA was demonstrably changed in the presence of AAP. The skeletal structure of BSA loosened, exposing internal hydrophobic aromatic ring amino acids and peptide strands to the solution.

Investigation on the toxic interaction of toluidine blue with calf thymus DNA

The gene toxic interactions of toluidine blue (TB) with calf thymus DNA (ct-DNA) were examined in vitro with UV-visible absorption spectroscopy, fluorescence spectroscopy, fluorescence polarization and circular dichroism techniques. The experimental results showed that TB interacted with ct-DNA by two binding modes. At low TB concentrations, TB intercalated into the DNA base pairs. At higher TB concentrations, TB was attached to the negatively charged phosphate groups. For the intercalation binding mode and electrostatic binding mode, the binding constants were 1.76 x1 0(6)L mol(-1) and 6.18 x 10(5)L mol(-1) and the number of binding sites was 0.48 and 0.79, respectively. Circular dichroism results showed that the two binding modes had different effects on the ct-DNA conformation. At high dye concentrations, Z-form DNA appears, while at low TB concentrations, a B to A form transition is observed.

Spectroscopic investigation on the toxic interactions of Ni2+ with bovine hemoglobin.

The toxic interaction between Ni(2+) and bovine hemoglobin (BHb) was investigated using fluorescence spectroscopy, synchronous fluorescence spectroscopy, ultraviolet-visible (UV-vis) absorption spectroscopy and circular dichroism spectroscopy (CD) under simulated physiological conditions. The experimental results showed that both dynamic and static quenching occurred simultaneously when Ni(2+) quenched the fluorescence of BHb. The binding site number n, apparent binding constant K(a) and corresponding thermodynamic parameters were measured at different temperatures. There was formation of Ni-BHb complex, but the binding between Ni(2+) and BHb was not strong. The process of the formation of Ni-BHb complex was a spontaneous interaction procedure in which electrostatic interaction played a major role. In addition, UV-vis and CD results showed that the addition of Ni(2+) changed the conformation of BHb.

Spectroscopic investigation on the toxicological interactions of 4-aminoantipyrine with bovine hemoglobin.

The effects of 4-aminoantipyrine (AAP) on bovine hemoglobin (BHb) were investigated by fluorescence spectroscopy, synchronous fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy and circular dichroism spectroscopy (CD) under simulated physiological conditions. The experimental results showed that AAP effectively quenched the intrinsic fluorescence of BHb via static quenching. The number of binding sites, the binding constant Ka, and the thermodynamic parameters (ΔH○, ΔS○ and ΔG○) were measured at two different temperatures. Van der Waals’ interactions and hydrogen bonds were the predominant intermolecular forces in stabilizing the BHb-AAP complex. The experiment results confirmed micro-environmental and conformational changes of BHb in the presence of AAP. The α-helix content decreased, indicating that AAP destroys some of the hydrogen bonding networks in the polypeptide chain.

Cyclic voltammetry: A new strategy for the evaluation of oxidative damage to bovine insulin

Research on protein oxidative damage may give insight into the nature of protein functions and pathological conditions. In this work, the oxidative damage of bovine insulin on Au electrode was investigated by cyclic voltammetry (CV). The experimental results show that there are two anodic peaks for the oxidative damage of bovine insulin, which arise from the oxidation of the exposed disulfide bond SSCYS7A,CYS7B, forming sulfenic acid RSOH (1.20 V, vs. SCE), sulfinic acid RSO2H and sulfonic acid RSO3H (1.35 V, vs. SCE). These in vitro findings not only demonstrate the applicability of CV in simulating/evaluating the oxidative damage of nonredox proteins but also find two promising candidates (two anodic peaks) for measuring insulin.

Spectroscopic investigation on the interaction of Cr(VI) with bovine serum albumin

The interaction of potassium dichromate (Cr(VI)) with bovine serum albumin (BSA) was investigated by fluorescence, synchronous fluorescence, resonance light scattering (RLS), ultraviolet–visible absorption, and circular dichroism (CD) spectroscopies under simulated physiological conditions. The experimental results showed that Cr(VI) could quench the intrinsic fluorescence of BSA following a static quenching process, which indicates the formation of a Cr(VI)–BSA complex. The binding constant (KA) and binding site (n) were measured at different temperatures. The spectroscopic results also revealed that the binding of Cr(VI) to BSA can lead to the loosening of the protein conformation and can change the microenvironment and skeleton of BSA.

A Unique Approach to the Mobile Proton Model: Influence of Charge Distribution on Peptide Fragmentation

The cleavage processes of protonated peptides in mass spectrometry, described in the mobile proton model, are charge-directed and depend on the charge distribution around the cleavage sites. Previous studies experimentally verified the mobile proton model by changing peptide sequences. In this study, oxidation was applied to change the charge distribution of peptides, but the sequence was retained. Tandem mass spectrometry (MS/MS) and quantum chemical calculations at the B3LYP/6-31G(d) level were used to test the validity of the mobile proton model. The results showed prominent differences of peptide fragmentation efficiency caused by the charge distribution produced by various oxidation levels. Fragmentation efficiency curves coupled with the relative intensities of the fragments indicated that the cleavage of the peptide Ala-Arg-Arg-Ala (ARRA) became more and more difficult as O atoms were added. The relative charge ratios between C and N atoms in the amide bonds decreased with the increase of oxidation extent, suggesting that oxidation resulted in protons moving away from the amide bonds. The combined methods proposed here provide a unique approach to substantiate and refine the mobile proton model for peptide fragmentation.

The oxidative products of methionine as site and content biomarkers for peptide oxidation

Biomarkers for peptide/protein oxidation under oxidative stress (OS) hold both incredible application potential as well as significant challenges. In this article, liquid chromatography and mass spectrometry were applied to establish a new method for evaluating the oxidation site and degree of peptide oxidized, with its oxidative product serving as biomarker. In the three model peptides, peptide FMRF (containing a methionine) was prone to undergo oxygen addition under UV/H2O2 oxidization, forming a sulfoxide (FM(O)RF) with a stable chromatographic peak separate from the model peptides. The oxidation content of FMRF, expressed as SFM(O)RF/(SFM(O)RF + SFMRF), is positively correlated with oxidation time. Based on sequence analysis of FM(O)RF, the oxidation mechanism (site and extent) of FMRF under UV/H2O2 oxidization was explicitly clarified. By comparing the specific injury to each model peptide, we found that the oxidative products of Met‐containing peptides are good biomarkers for OS. This research not only expands the range of biomarkers for OS, but also provides an efficient and accurate method for evaluating oxidation damage to peptides and even proteins. Copyright

A new biomarker of protein oxidation degree and site using angiotensin as the target by MS

Hydroxyl radicals generated from Fenton reaction were used to damage the angiotensin. The oxidative damage degree and sites of peptides were measured by HPLC-MS and MS/MS. Experimental results proved that the oxidative damage degree increased with longer reaction time. The results also showed that the side chains of phenylalanine and tyrosine in angiotension can be attacked by hydroxyl radicals to form the oxidative products. A new strategy was established to monitor the oxidative degree and sites of peptides and laid the foundation for protein oxidation. This method can be used to investigate the mechanism of protein oxidative damage caused by oxidative stress which is induced by environmental pollutants and physiological activities. There will also be a wide application in the research of pathogenesis of some disease related to oxidative stress.

Influence of charge distribution on the discrepant MS/MS fragmentation of the native and oxidized FMRF: evidence for the mobile proton model

Using the mobile proton model as a framework, the influence of charge distribution on the discrepant fragmentation of peptides FMRF, FM(O)RF and FM(O2)RF (with united peptide sequence) was explored by mass spectrometry experiments and quantum chemical calculations. With the added O atoms, more negative charges were prompted to deposit in the main protonation sites of the oxidation products. Consequently, the solvated proton to the oxidized peptides could flow to the amide bonds in an easier manner and made these bonds fragment easily. Oxidation also induced the discrepant fragmentation of these bonds in a predictable manner: the more negative charges deposited in an amide bond, the more daughter ions (an, bn, yn ions and their derivatives) were produced. The combined methods proposed here refined the mobile proton model for peptide fragmentation and opened the way to probe the discrepant fragmentation of peptides in peptide/protein identification. Copyright