Robert Gradinaru

Interaction of inorganic mercury with CoA-SH and acyl-CoAs

Sulfur containing biomolecules are involved in complexes with mercury. CoA is an important cofactor for many enzymes involved in metabolic processes. Fatty acyl-CoA-thioesters, substrates of mitochondrial ß-oxidation, are sulfur containing compounds and potential mercury ligands. The CoA-Hg2+ complex can be easily assessed by UV-Vis spectroscopy or indirectly by antibacterial tests that reconfirmed the protective role of CoA on E. coli. The characteristics of these complexes were determined by means of FTIR spectroscopy. The reverse phase liquid chromatography combined with electrospray ionization tandem mass spectrometry was used for detection of the side-product that resulted through the cleavage of thioesters in the presence of mercury. An unexpected result was the detection of octathioic acid as a product. Our study shows that mitochondrial β-oxidation can be affected by thioesters depletion assisted by Hg2+. The GC-MS technique could be used to detect some possible mitochondrial injuries due to the heavy metal ions.

Ultrasound-based protein determination in maize seeds

The need for a simple and accurate method for protein estimation in alcoholic extracts led to the reexamination of the optimum conditions of a colorimetric assay based on the biuret reaction. Sonication time and the other experimental parameters were optimized after kinetics study on the extraction of either zein or total proteins. Zein extraction and purity were investigated by (1)H and (13)C NMR spectroscopy, SDS-PAGE electrophoresis, and UV-visible spectrophotometry (UV-vis). A zein assay was proposed, which involves the reaction of copper ions in copper phosphate powder with zein extracted in ethanolic solutions under strong alkaline environment. Furthermore, we extended this procedure to determine total proteins in maize samples simultaneously with their ultrasonic-assisted (US) extraction with an alkaline-alcoholic solution. Proteins in both types of extracts were well characterized by UV-vis spectroscopy. However, the 545 nm absorbance of the violet-colored supernatants which is proportional to the protein content was found to be the key parameter of the improved biuret-based protein assay. Comparison of values obtained by this procedure and by Micro-Kjeldahl method was in excellent agreement. A scaled-down procedure agreed well with the standard procedure. Enhanced accuracy and repeatability was found in protein determination in maize using the modified biuret method. The optimization of reagent concentrations and incubation times were studied as well.

Letter: Study on the mechanism of ferrite-induced dinitrophenol photodegradation

We report here the effectiveness of gas chromatography mass spectrometry techniques in establishing the ferrite-associated photocatalytic degradation mechanism of pesticide 2,4-dinitrophenol (2,4-DNP). Unlike the previously discussed DNP-degradation mechanisms that involve either oxidation or reduction reactions, ferrite-based ultraviolet (UV) photodegradation of DNP affords the nontoxic 6-hydroxy-3,5-dinitrohexa-2,4-dienal by an unusual water addition to the benzene core. We searched for and demonstrated the presence of an epoxide of DNP within the photodegradation process, which may unambiguously explain the novel photochemical mechanism. During the 15 min UV photoinduced process, DNP degradation efficiency on the zinc ferrite catalyst was calculated to be 82%, whereas the first-order kinetic rate constant k was as high as 3.4 × 10−2 min−1.

Ninhydrin-based spectrophotometric assays of trace cyanide

Abstract The extreme toxicity of cyanide, its wide industrial application as well as its continued illegal use generate research interest in different fields of science, imposing multidisciplinary approach to study cyanide poisoning. We show here that the reaction between cyanide and ninhydrin can be performed at ambient conditions; however, the ninhydrin reagent has to be freshly prepared in oxygen free solvent. Besides, we show that the reading of the absorbance at 485 nm might be more suitable and reliable than that at 590 nm, where the pH-dependent blue colored cyanide-ninhydrin adduct is less stable. Ninhydrin-based color reagent can be used to quantify the cyanide released from plant seeds. In sodium carbonate medium, the proposed assay is fast, cheap and environmentally friendly

Letter: Mass Spectrometric Evidence for Iron Binding to the Neuroprotective Peptide NAP and its Cys5 Mutant:

The NAP peptide (H2N-1NAPVSIPQ8-CONH2) is a truncated version of the activity-dependent neuroprotective protein. Its neuroprotective activities consist of the inhibition of Aβ(25–35) and Aβ(1–40) fibrillogenesis as well as protection against Aβ-induced neurotoxicity and prevention of microtubule disruption associated with Alzheimers disease. Therefore, we synthesized NAP and its mutant peptide with the sequence H2N-1NAPVCIPQ8-COOH (NAPCOH) by replacing serine S5 with cysteine C5. Both native and mutant peptides were further used to study their interaction with iron ions. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry, Fourier transform infrared spectroscopy and also atomic force microscopy were used to probe Fe3+ binding to both peptides. Contrary to the expected results, the investigated peptides underwent different oxidation processes, with resultant reduced Fe2+ ions. These ions, and not the original Fe3+ ions, were found to bind to each of the non-oxidized peptides.

Banned dinitrophenols still trigger both legal and forensic issues

ABSTRACT Numerous hazardous chemicals from various industrial sources enter the environment daily; some of them can be used both as drugs and as toxins. Many of these compounds, including dinitrophenols, such as 2,4-dinitrophenol (2,4-DNP), 4,6-dinitrocresol (DNOC), and 2-sec-butyl-4,6-dinitrophenol (dinoseb), are widely used pesticides that persist in some contaminated soils. They have been found in groundwater, causing health and environmental hazards, and are subjects of forensic toxicology. Dinitrophenols have multiple biological and noxious effects based on a mechanism of action characteristic to metabolic inhibitors. Although banned, they have pharmaceutical activity and can be purchased on the Internet as ingredients of weight-loss pills. Many death cases have been reported as accidents in agriculture or as overdoses in weight-loss diets. In this article, we discuss legal aspects of dinitrophenol usage, as well as their biological effect and possible mechanisms of toxicity.

Interaction of Amyloid Aβ(9–16) Peptide Fragment with Metal Ions: CD, FT-IR, and Fluorescence Spectroscopic Studies

Heavy metal ions and those of aluminum may interact with amyloid-β peptides (Αβ) associated with Alzheimer’s disease, contributing to Aβ aggregation and fibrillation that worsen this neuropathology. Nevertheless, the precise residues involved in metal ligation or interaction are yet to be established, although the N-terminal Aβ(1–16) peptide fragment is considered to be the metal-binding domain. However, because the results of the metal ion binding studies using Aβ(1–16) were not very conclusive, we have investigated shorter peptides. Here, we report the synthesis of truncated Aβ(9–16) peptide of the human Aβ(1–40) and Aβ(1–42) peptides, as well as its characterization by nuclear magnetic resonance and mass spectrometry. Furthermore, the Aβ(9–16) peptide interactions with various metal ions like Cu2+, Zn2+, Fe3+, Al3+, and Ni2+ were studied by circular dichroism, Fourier transform-infrared, and fluorescence spectroscopy. Our results show that the newly synthesized peptide sequence, which contains just two histidine and one tyrosine residues, seems to be a key site for metal binding, which was not thoroughly investigated so far. Light scattering spectra of Aβ(9–16) and its complexes with metal ions were measured, whereas some fluorescence experiments were also performed. Our data suggest that Aβ(9–16) peptide fragment coordination by metal ions is dependent on the type of metal, and the amino acid residues involved in metal bonding.Graphical Abstract

Improved ninhydrin-based reagent for spectrophotometric determination of ppb levels of cyanide

ABSTRACT Cyanide is an extremely severe poison that has been raising concerns and controversies regarding its toxic effects on humans and the environment. A short-term human exposure to elevated levels can induce coma or death. Toxic effects occur at blood cyanide concentrations of 0.05 mg/dL, while death can occur at levels of 0.3 mg/dL. Numerous law and systems regulate cyanide levels in various environments. To exemplify, the maximum contamination limit imposed by United States Environmental Protection Agency in drinking water is 200 µg/L, while the European Union presents a lower limit of 50 µg/L. Ninhydrin has proved to be an effective color reagent for the spectrophotometric determination of trace levels of cyanide. This article reports a ninhydrin-based color reagent that was employed in cyanide determination involving real samples of water, cigarette smoke, urine, and cyanogenic plants, achieving an appropriate sample pretreatment procedure for cyanide liberation and separation. The reaction between cyanide and ninhydrin is fast enough to be performed at ambient conditions, but the ninhydrin reagent must be freshly prepared in oxygen-free solvent. Beers law is obeyed in the range between 20 and 400 ppb CN− (r = 0.992; N = 7) with molar absorptivity of 1.4 × 105 L mol−1 cm−1 and limit of detection of 8 ppb cyanide. Recovery at 100 ppb CN− was (100.5 ± 0.1) %, whereas precision was 1.7%. Also, absorbance at 485 nm proved to be temperature-dependent, with a maximum at ambient conditions. Throughout all measurements, the ninhydrin-based assay proved to be reliable, economical and environmentally friendly. Significance of the assay for the determination of cyanide in the field of environmental forensics, as well as its potential aid in elucidating incidents with legal consequences are highlighted.

New insights into coenzyme A interaction with mercury ions provided by mass spectrometric and circular dichroism spectroscopic approaches

The interaction of coenzyme A (CoA) with mercury ions was investigated using electrospray ionization mass spectrometry and circular dichroism spectroscopy. Our results indicated a 1:1 stoichiometric CoA–Hg complex at physiological pH. Furthermore, the CoA conformation considerably changed in the presence of mercury ions. In addition, a by-product of the reaction, thiocoenzyme A, was identified using mass spectrometry.