Emilia Bramanti

Determination of the Relative Amount of Nucleic Acids and Proteins in Leukemic and Normal Lymphocytes by Means of Fourier Transform Infrared Microspectroscopy

Previous Fourier transform infrared (FT-IR) spectroscopic studies on neoplastic and normal cells have shown different band profiles and intensity associated with absorptions of proteins and nucleic acids. In the present study, an interpretation of such differences has been attempted by comparing the spectra of DNA/RNA/protein mixtures with the spectra, particularly, obtained for lymphocytes from B-chronic lymphatic leukemia (B-CLL) patients and normal donors. FT-IR microspectroscopy analysis showed a good agreement between the intensity and the band profile of the spectra of leukemic lymphocytes and those of the binary mixture made up of 75% human serum albumin and 25% DNA. The addition of small amounts of RNA (1–5%) modified the band shape, making it more similar to the spectrum of normal lymphocytes. An attempt was also made to estimate the relative amounts of DNA and RNA. The results demonstrated an increase in the DNA/RNA ratio value in neoplastic lymphocytes with respect to that reported in literature for normal ones.

Determination of the secondary structure of isomeric forms of human serum albumin by a particular frequency deconvolution procedure applied to fourier transform IR analysis

A new deconvolution procedure was applied to the analysis of Fourier transform ir spectra of human serum albumin secondary structure in the native state and in states denatured by heat and acid treatment. The deconvolution method is based on the use of the Conjugate Gradient Minimization Algorithm, with the addition of suitable constraints directly obtained by the application to the measured spectrum of the second derivative operator. This method computes central band frequency, bandwidth, and amplitude of the different spectral components of conformation-sensitive amide bands. In the specific case, it was applied to analysis of the amide I band, and the quantitative determination of the different secondary structures (alpha-helix, beta-sheet, beta-turns, and random) was attempted for all the samples examined. The precision of the quantitative determination depends on the amounts of these structures present in the protein. The coefficient of variation is < 10% for values of amide I component > 15%. The accuracy was tested by comparing, by means of linear regression, the results obtained for human serum albumin, hemoglobin, alpha-chymotrypsin, and cytochrome c, using our method, with those obtained by x-ray crystallography and CD; the results obtained by other vibrational spectroscopic approaches were also compared. The fit standard error between x-ray and ir secondary structure values estimated by our method is 2.5% for alpha-helix, 7.16% for beta structures, and 5.1% for other structures (turns and random coils). Quantitative results are given for the secondary structures (alpha-helix, turns, and beta-strands) present in the native state (turns and beta-strands up to now unknown in aqueous solution), together with the percentages of these structures and additional ones (random coils and beta-sheets) formed during denaturization.

Separation and determination of denatured αs1-, αs2-, β- and κ-caseins by hydrophobic interaction chromatography in cows’, ewes’ and goats’ milk, milk mixtures and cheeses

Abstract Caseins α s1 -, α s2 -, β- and κ- from raw cows’, ewes’ and goats’ milk were separated and determined by hydrophobic interaction chromatography (HIC) by using a Propyl column (Eichrom) in the presence of 8.0 M urea in the mobile phase. The method is based on fast and easy solubilization of real raw samples by 4.0 M guanidine thiocyanate followed by the HIC analysis, without any preliminary precipitation or separation of the casein fraction. Elution conditions have been optimized by analyzing commercial single bovine standard caseins and their mixture. In the optimized chromatographic conditions the four casein fractions were separated in less than 45 min. A linear relationship between the concentration of casein and peak area (UV absorbance detector at 280 nm) has been obtained over the concentration range of 0.5 to 40 μ M . The detection limit for α-, β- and κ-caseins ranged between 0.35 and 0.70 μ M . The precision of the method was evaluated, the coefficient of variation for α-, β- and κ-casein determination ranging between 3.0 and 6.0%. The method has been validated by the analysis of reference skim milk powder (BCR-063R) certificated for total nitrogen content. The method was applied to commercial casein mixture and to the qualitative and quantitative analysis of casein fractions in unprocessed, raw cows’, goats’ and ewes’ milk (10 samples analyzed for each species), in one sample of unprocessed buffalos’ milk and in commercial cheeses (mozzarella, robiola, ricotta and stracchino). Binary mixtures of milk (cow/goat and cow/ewe) were also analyzed and the ratio between casein peak areas (α s1 /κ, α s2 /β, β/κ and α s2 /α s1 ) of the HIC chromatograms was proposed and discussed in order to evaluate a possible application of this method to detect milk adulteration.

Short-time effect of heavy metals upon microbial community activity

Microcalorimetry was applied to assess and compare the toxic effect of heavy metals, such as As, Cu, Cd, Cr, Co, Pb and Zn, on the soil microbial activities and community. About 1.0 g soil spiked 5.0mg glucose and 5.0mg ammonium sulfate, the microbial activities were recorded as power-time curves, and their indices, microbial growth rate constant k, total heat evolution Q(T), metabolic enthalpy Delta H(met) and mass specific heat rate J(Q/S), were calculated. Comparing these thermodynamic parameters associated with growth yield, a general order of toxicity to the soil was found to be Cr>Pb>As>Co>Zn>Cd>Cu. When soil was exposed to heavy metals, the amount of bacteria and fungi decreased with the incubation time, and the bacterial number diminished sharply. It illustrates that fungi are more tolerant, and bacteria-fungi ratio would be altered under metal stress. To determine the status of the glucose consumed, a glucose biosensor with eggshell membrane was used to measure the remaining glucose in soil sample. Results showed that the time at which glucose was consumed completely was agreed with the microcalorimetric time to a large extent, and depended on the toxicity of heavy metals as well.

Mercury speciation by liquid chromatography coupled with on-line chemical vapour generation and atomic fluorescence spectrometric detection (LC-CVGAFS)

Reverse phase chromatography (RPC) coupled on-line with UV-vis diode array detector (DAD) and cold vapour generation atomic fluorescence spectrometry (CVGAFS) is proposed for the speciation and determination of inorganic and organic mercury (methylmercury, ethylmercury and phenylmercury) in the form of cysteine, penicillamine and glutathione complexes. The mercury-thiol complexes are separated on a C(18) Reverse Phase column and oxidized on-line with bromine, generated in situ by KBr/KBrO(3) in HCl medium, in order to fast convert organic mercury species to inorganic mercury in less than 2.5s, at room temperature, in a 30cm knitted coil. Hg(II) is selectively detected by AFS in a Ar/H(2) miniaturized flame after sodium borohydride reduction to Hg(0). Under optimized conditions, on-line bromine treatment gives recoveries of thiol-complexed methylmercury, ethylmercury and phenylmercury with respect to inorganic mercury ranging between 79 and 85%, 80 and 85%, 63 and 76%, respectively, depending on the complexing thiol employed. Optimized elution conditions were provided in the three complexing agents. The detection limits (LODc) for inorganic mercury, methylmercury, ethylmercury and phenylmercury, in the optimized conditions complexed with thiols were about 16, 18, 18 and 20pg (as mercury), respectively, a relative standard deviation (R.S.D) ranging between 1.5 and 2.0%, and a linear dynamic range between 0.1 and 100ng injected. LC-DAD-CVGAFS method has been validated by analysing two certificate reference material, DORM-2 and NIES CRM 13, obtaining 98+/-6 and 97+/-5% of methylmercury recovered, respectively.

Study on the toxic effects of diphenol compounds on soil microbial activity by a combination of methods

Microcalorimetric technique based on heat-output measurement, direct microorganism counting and enzymatic activity determination, have been explored to evaluate the toxic effects of diphenol species (catechol, resorcinol, and hydroquinone) on soil microbial activity. The thermokinetic parameters including growth rate constant (k), inhibitory ratio, half inhibitory concentration and total thermal effect (Q(total)), were calculated and compared using the data obtained from the power-time curves of the microcalorimeter. It was found that addition of high concentrations of diphenol compounds to the soil samples resulted in low microorganism counts. The trend of the number of cultivable microorganisms with increasing concentration of diphenols was similar to specific growth rate k. It appeared that the higher the water soluble carbon (WSC) content, the higher the Q(total) value. The low dehydrogenase and beta-glucosidase found in the soils treated by catechol and hydroquinone was possibly due to their low WSC concentration and high inhibitory effects, respectively. The results reveal the toxicity of the three diphenols in a descending sequence: hydroquinone, resorcinol and catechol. The combination of the three methods is a more comprehensive toxicological investigation of a complex microbiological system. Microcalorimetry is for studying the metabolic growth of microorganisms, the plate counting method is for quantifying the real microbial growth, and the soil enzyme activity is for assessing the intracellular and extracellular activity of microbial biomass. Our proposed methods can provide toxicological information of diphenols to soil microbes from the metabolic, microbial and biochemical point of views which are consistent with and correlated to each other.

Cold vapour atomic fluorescence studies on the behaviour of mercury(II) and mercury(II)-thiol complexes. An alternative route for characterization of –SH binding groups

The behaviour of Hg II and Hg II -thiol complexes (RSH=L-cysteine, DL-penicillamine, propane-2-thiol, glutathione, thiosalicylic acid) following their reduction with alkaline sodium tetrahydroborate to give Hg 0 has been studied by using a continuous flow reaction system coupled with atomic fluorescence spectrometric (AFS) detection. The quantitative reduction of Hg II to Hg 0 takes place with a specific amount of sodium tetrahydroborate according to the stoichiometric reaction of mercury with alkaline NaBH 4 . The complete reduction of Hg II -thiol complexes to Hg 0 requires a molar excess of NaBH 4 of up to six orders of magnitude, depending on the type of complex. Under an appropriate excess of reductant, Hg II and its thiol complexes are not distinguishable giving the same AF molar response. The method allows the discrimination of Hg II from Hg II -thiol complexes without any preliminary separation. Applications to the indirect titration of thiols and to the determination of the number of accessible }}n1SH groups in pure ovalbumin samples are reported.

Physico-chemical characterization of protein–pigment interactions in tempera paint reconstructions: casein/cinnabar and albumin/cinnabar

AbstractIn this work, we characterized paint reconstructions using ovalbumin and casein as binders, and cinnabar (HgS) as a pigment, before and after artificial ageing. Egg and casein are common paint binders that were used historically in the technique of tempera painting. Despite extensive research on the identification of proteinaceous binders in paintings, there is a substantial lack of knowledge regarding the ageing pathway of their protein content, and their chemical interaction with inorganic pigments. Thermogravimetric analysis, infrared spectroscopy and size-exclusion chromatography (SEC) were used to reveal the physico-chemical processes involved in the ageing of proteins in paintings. Taken together, the three techniques highlighted that proteins are subject to both cross-linking and hydrolysis upon ageing, and to a lesser extent, to oxidation of the side chains. Mercury–protein interactions were also revealed using a cold vapour generation atomic fluorescence spectrometer mercury-specific detector coupled to SEC. The study clearly showed that HgS forms stable complexes with proteins and acts as a sensitizer in cross-linking, hydrolysis and oxidation. FigureA multi-techinque approach to the study of protein/cinnabar tempera paint recontructions: thermogravimetric analysis, Fourier Transform Infrared Spectroscopy and size exclusion chromatography

Determination of copper complexation in sea water by a ligand competition technique with voltammetric measurement of the labile metal fraction

Abstract A ligand competition—anodic stripping voltammetric (ASV) technique is proposed for investigating copper complexation in sea water. Increasing amounts of ethylenediamine were used to convert the non-reducible copper fraction into a labile copper complex and the change in the stripping peak current was followed. Ligand titration of sea water using this technique indicated that the naturally occurring non-labile copper fraction is bound to ligands having concentrations less than 5 × 10 −9 M and a conditional stability constant (log K ′ CuL ) of 13.1 ± 0.3. An advantage of ligand exchange voltammetry is that when applied to raw sea water, it allows the investigation of copper ‘already” bound by inert ligands. On the other hand, when applied to copper-spiked sea-water samples, this method permits the investigation of the “newly formed” inert complexes. It was found that when copper was added to give [Cu] > × 10 −8 M, the added copper assumed a different inert form than that of the already bound copper in the sample. The combined use of ligand competition—ASV with stripping polarography as a diagnostic method of speciation analysis is discussed.

Microwave-assisted photochemical reactor for the online oxidative decomposition and determination of p-hydroxymercurybenzoate and its thiolic complexes by cold vapor generation atomic fluorescence detection.

We developed a photochemical method for the online oxidation of p-hydroxymercurybenzoate (PHMB), an organic mercury species widely used for mercaptan and thiolic compound labeling. The method is based on a fully integrated online UV/microwave (MW) photochemical reactor for the digestion of PHMB, followed by cold vapor generation atomic fluorescence spectrometry (CVG-AFS) detection. The MW/UV process led to the quantitative conversion of PHMB and thiol-PHMB complexes to Hg(II), with a yield between 91% and 98%, without using chemical oxidizing reagents and avoiding the use of toxic carcinogenic compounds. This reaction was followed by the reduction of Hg(II) to Hg(0), performed in a knitted reaction coil with NaBH(4) solution, and AFS detection in an Ar/H(2) miniaturized flame. The low MW power applied (18 W) allowed us to keep constant the temperature of the photochemical reactor (21 ± 1 °C), using a flowing water bath. This avoided peak widening due to diffusion processes generally occurring at high temperatures and in the additional cooling coil. This method has been applied to the determination of thiols in human plasma, blood, and wine.