Tryptophan Emission Spectra Research Articles

Fluorescence Spectroscopy Based Identification of Pseudomonas Aeruginosa and Escherichia Coli Suspensions.

In this article, Fluorescence spectroscopy has been employed for the identification of Pseudomonas aeruginosa (PA) and Escherichia coli (E. coli) in water suspension. Emission spectra of PA and E. coli suspensions have been acquired by using excitation wavelengths from 270 to 420nm with steps of 10nm to explore their spectral features. It has been found that the emission spectra of tryptophan, tyrosine, NADH and FAD, being the intracellular biomolecules present in both bacteria, can be used as fingerprints for their identification, differentiation and quantification. Both bacterial strains can clearly be differentiated from water and from each other by using λex 270-290nm through spectral analysis and from λex: 300-500nm by applying statistical analysis. Furthermore, calibration curves for different bacterial loads of PA and E. coli suspensions have been produced between colonies forming units per ml (CFUs/ml) the integrated intensities of their emission spectra. CFUs/ml of both bacterial suspensions have been determined through plate count method which was used as cross-reference for the analysis of emission spectra of both bacterial suspensions. These curves may be used to estimate CFU/ml of both PA and E. coli in unknown water suspensions by determining the integrating intensity of their emission spectra.

Fluorescence Spectroscopy Based Characterization of Pseudomonas Aeruginosa Suspension.

In this article, optical characterization of Pseudomonas aeruginosa (PA) suspension has been performed by using Fluorescence spectroscopy. Optical density (OD) and plate count methods have been employed as a reference for the analysis of emission spectra of Pseudomonas aeruginosa in water suspension. Emission spectra of PA suspension has been acquired by using excitation wavelengths from 270 to 420nm with step of 10nm to explore its spectral behavior. It has been found that emission spectra of tryptophan, tyrosine, NADH and FAD, the intracellular biomolecules of bacteria, can be used as finger prints for the detection of Pseudomonas aeruginosa. Furthermore, the effect of water matrix on the spectral emission of Pseudomonas aeruginosa has been investigated that might be one of the limitation of Fluorescence spectroscopy for complex water matrices. Moreover, a calibration curve has been produced between ODs600 of Pseudomonas aeruginosa suspensions of different bacterial load and integrated intensities of the emission spectra of same samples. These ODs600 and integrating intensities have been further vetted through plate count method by determining their corresponding colony forming units per ml (CFU/ml). This calibration curve may be used to determine CFU/ml of Pseudomonas aeruginosa in water sample by determining integrating intensity of its emission spectrum.

Calcium dependence of both lobes of calmodulin is involved in binding to a cytoplasmic domain of SK channels.

KCa2.1-3 Ca2+-activated K+-channels (SK) require calmodulin to gate in response to cellular Ca2+. A model for SK gating proposes that the N-terminal domain (N-lobe) of calmodulin is required for activation, but an immobile C-terminal domain (C-lobe) has constitutive, Ca2+-independent binding. Although structures support a domain-driven hypothesis of SK gate activation by calmodulin, only a partial understanding is possible without measuring both channel activity and protein binding. We measured SK2 (KCa2.2) activity using inside-out patch recordings. Currents from calmodulin-disrupted SK2 channels can be restored with exogenously applied calmodulin. We find that SK2 activity only approaches full activation with full-length calmodulin with both an N- and a C-lobe. We measured calmodulin binding to a C-terminal SK peptide (SKp) using both composition-gradient multi-angle light-scattering and tryptophan emission spectra. Isolated lobes bind to SKp with high affinity, but isolated lobes do not rescue SK2 activity. Consistent with earlier models, N-lobe binding to SKp is stronger in Ca2+, and C-lobe-binding affinity is strong independent of Ca2+. However, a native tryptophan in SKp is sensitive to Ca2+ binding to both the N- and C-lobes of calmodulin at Ca2+ concentrations that activate SK2, demonstrating that the C-lobe interaction with SKp changes with Ca2+. Our peptide-binding data and electrophysiology show that SK gating models need deeper scrutiny. We suggest that the Ca2+-dependent associations of both lobes of calmodulin to SKp are crucial events during gating. Additional investigations are necessary to complete a mechanistic gating model consistent with binding, physiology, and structure.

A rapid method to quantify casein in fluid milk by front-face fluorescence spectroscopy combined with chemometrics

Casein in fluid milk determines cheese yield and affects cheese quality. Traditional methods of measuring casein in milk involve lengthy sample preparations with labor-intensive nitrogen-based protein quantifications. The objective of this study was to quantify casein in fluid milk with different casein-to-crude-protein ratios using front-face fluorescence spectroscopy (FFFS) and chemometrics. We constructed calibration samples by mixing microfiltration and ultrafiltration retentate and permeate in different ratios to obtain different casein concentrations and casein-to-crude-protein ratios. We developed partial least squares regression and elastic net regression models for casein prediction in fluid milk using FFFS tryptophan emission spectra and reference casein contents. We used a set of 20 validation samples (including raw, skim, and ultrafiltered milk) to optimize and validate model performance. We externally tested another independent set of 20 test samples (including raw, skim, and ultrafiltered milk) by root mean square error of prediction (RMSEP), residual prediction deviation (RPD), and relative prediction error (RPE). The RMSEP for casein content quantification in raw, skim, and ultrafiltered milk ranged from 0.12 to 0.13%, and the RPD ranged from 3.2 to 3.4. The externally validated error of prediction was comparable to the existing rapid method and showed practical model performance for quality-control purposes. This FFFS-based method can be implemented as a routine quality-control tool in the dairy industry, providing rapid quantification of casein content in fluid milk intended for cheese manufacturing.

Utilization of front-face fluorescence spectroscopy for monitoring lipid oxidation during Lebanese Qishta aging

Front-face fluorescence spectroscopy technique coupled with chemometric tools was used for predicting the freshness state of a Lebanese dairy product called Qishta, stored up to 20 days. Acid, peroxide and thiobarbituric acid reactive substances (TBARS) values reached no more than 0.93 mg NaOH g−1 fat, 6.22 meq O2 Kg−1 fat, and 0.0313 mg malonaldehyde (MA) kg−1 sample, respectively, throughout the investigated storage time. In parallel, fluorescence emission spectra of tryptophan and riboflavin, and fluorescence excitation spectra of vitamin A were recorded and showed the highest fluorescence intensity for the Qishta samples aged of 20 days and the lowest intensity for the fresh ones. The primary and secondary indicators of lipid oxidation showed that Qishta can be stored for 20 days without any alteration despite the increase in the TBARS after 16 days of storage. Principal component analysis (PCA) applied on riboflavin emission spectra allowed better discrimination between Qishta samples with a clear distinction of those aged 20 days while some overlapping was noticed between samples aged below 16 days. A high correlation (R2 = 0.923) was observed between the peroxide value and the intensity of the riboflavin fluorescence recorded at 460 nm.

Both Lobes of Calmodulin Bound to KCa2.2 Respond to Ca2+

An increase in intracellular Ca2+ opens KCa2.2 Ca2+-activated K+-channels (SK) to conduct K+ down its electrochemical gradient. SK activation requires bound calmodulin proteins to decode dynamic Ca2+ fluctuations in a cell. Bilobal calmodulin has four Ca2+ binding sites. A common model proposes that just the N-terminal domain (N-lobe) of calmodulin binds Ca2+ to open SK, while an immobile C-terminal domain (C-lobe) has constitutive, Ca2+-independent binding. Such a mechanism is compatible with x-ray and EM structures of intact SK channels and fragments. While molecular structures provide snapshots of the channels in different conformations with and without calcium, gating is a dynamic process involving several different conformations, calcium binding to calmodulin, interactions between calmodulin and the channel and channel opening. We measured SK activity using inside-out patch recordings from “calmodulin-disrupted” SK channels where calcium-dependent activation can be restored by exogenously applied full-length calmodulin. Isolated N and C lobes have little ability to activate SK. We measured calmodulin binding to a C-terminal SK2 peptide (SKp) using both composition-gradient multi-angle light scattering and tryptophan emission spectra. While they do not rescue activation, isolated lobes do bind to a SK cytoplasmic domain with high affinity. Consistent with earlier models, N-lobe binding to SKp is stronger in Ca2+, and C-lobe binding affinity is strong regardless of Ca2+. However, a native tryptophan in SKp is sensitive to Ca2+ binding to either the N- or C-lobe of calmodulin at Ca2+ concentrations that activate SK. Our results show that the calmodulin C-lobe bound to SK is Ca2+ sensitive and that both lobes of calmodulin bind SK in Ca2+-dependent states governed by the different properties of each lobe.

Study of moisture content and water activity of rice using fluorescence spectroscopy and multivariate analysis.

The potential of fluorescence spectroscopy for non-destructive and rapid measurement of moisture content (MC) and water activity (aW) of rice was examined. A total of n = 15 samples of rice produced at different locations were studied. The emission fluorescence spectra of tryptophan (305-600 nm, excitation: 290 nm) and emission fluorescence spectra of riboflavin (410-700 nm, excitation: 370 nm) were collected from milled rice. MC and aW of the rice samples were measured. Spectral data were subjected to principal component analysis (PCA) and partial least squares discriminant analysis (PLSDA). PCA and PLSDA showed the rice samples can be differentiated according to their MC and geographical origin of cultivation. Partial least squares regression (PLSR) models obtained from tryptophan emission spectra predicted MC and aW with R2 = 0.82 and RMSEV = 0.427, and R2 = 0.74 and RMSEV = 0.512, respectively. PLSR models obtained using riboflavin emission spectra allowed predicting MC aW with R2 = 0.85 and RMSEV = 0.393, and R2 = 0.79 and RMSEV = 0.463, respectively. This study demonstrates that fluorescence spectroscopy combined with multivariate statistical tools has promising potential for non-destructive and rapid measurement of moisture content and water activity of rice.

Multifactors accelerated aging of sterilized acidic pudding determined by traditional and fluorescence techniques

AbstractThe aim of this study was to reproduce quality changes occurring in shelf‐stable pudding over 368 days, in less than 100 days by using one or more accelerating factors. The effect of headspace volume, packaging permeability, combination of packaging permeability and light, iron, and heat treatment on the evolution of pudding quality during storage was studied at the molecular and macroscopic levels. For control samples, an increase in the consistency, apparent viscosity, hysteresis area, G'‐G" crossover and yellowness was observed throughout storage. The rheology, pH, and colorimetric measurements failed to discriminate samples subjected to different accelerating factors from the control ones. Regarding the shapes of tryptophan and riboflavin emission spectra, samples with high permeability to oxygen and exposed to light were the most deteriorated, followed by those supplemented with iron or subjected to high heat treatment. However, iron and heat treatment trials were found to be the most suitable parameters to accelerate aging of pudding. Finally, both, headspace volume or packaging permeability did not affect the aging of acidic pudding samples.Practical applicationsSevere heat treatment or the addition of iron in the pudding recipe could be used as suitable parameters to accelerate shelf‐stable acidic pudding aging. Fluorescence spectroscopy technique showed a high discrimination power and can be applied for online monitoring of shelf‐stable dairy desserts quality, as it requires no preparation of the sample.

Quality Assurance of Model Infant Milk Formula Using a Front-Face Fluorescence Process Analytical Tool

Front-face fluorescence spectroscopy (FFFS) was evaluated as a quality assurance process analytical technology (PAT) tool for infant milk formula (IMF) manufacture. Batches of first-stage IMF (60:40 whey protein:casein ratio) powder were produced with protein:fat:lactose ratios of 1.3:3.6:7.3, differing only in heat treatment applied prior to spray drying (72, 95, or 115 °C for 15 s). Each IMF powder was stored at 15 ± 2 °C and 37 ± 2 °C and analyzed at months 0, 3, 6, and 12. Partial least squares (PLS) models were developed for IMF in both powder and liquid states using FFFS spectra to predict pre-drying heat treatment temperature, soluble protein content, and storage time. Models developed using tryptophan emission spectra for IMF powder predicted storage time, pre-drying heat treatment temperature, and soluble protein content with RMSECV values of 0.3 months, 8.3 °C, and 1.01 g protein/100 g powder, respectively. IMF powders were rehydrated to 13% total solids and analyzed using the vitamin A emission spectra. Models developed for rehydrated IMF predicted storage time and pre-drying heat treatment temperature with RMSECV values of 1.5 months and 6.7 °C, respectively. Surface free fats were predicted with an RMSECV range of 0.12–0.20% (w/w of powder) in rehydrated IMF. PLS discriminant analysis models developed for both powder and liquid IMF samples successfully discriminated for storage temperature. This preliminary study demonstrates the strong potential of FFFS as a PAT tool for IMF quality assurance.

Impact of Temperature Cycling and Isothermal Storage on the Quality of Acidic and Neutral Shelf-Stable Dairy Desserts Packaged in Flexible Pouches

The aims of the present study were, first, to identify the quality changes occurring in sterilized vanilla and strawberry pudding during storage at 20 °C; second, to understand the effect of storage temperature on aging; and, third, to determine if temperature could be used as an accelerating factor for shelf life or stability studies. Shelf-stable pudding was produced industrially and packaged in flexible pouches. Different ingredients were used to produce strawberry and vanilla pudding, and the resulting pH was 4.1 and 6.5, respectively. Pudding pouches were kept for 7 days at 20 °C and stored for up to 112 days at − 18, 4, 20, and 30 °C, or cycled repeatedly between − 18 and 20 °C, 4 and 20 °C, and 30 and 20 °C with a frequency of two cycles per week. Color, pH, apparent viscosity, flow behavior, and fluorescence spectra measurements were conducted up to 112 days of storage. The most relevant indicators to monitor quality changes in vanilla pudding were η, pH, b*, and tryptophan emission spectra, and in strawberry pudding, a*, b*, and riboflavin, tryptophan, and vitamin A emission spectra. Indeed, the temperature of 30 °C was identified as the most suitable accelerating factor for accelerated aging tests regarding changes in pH, color, riboflavin, and tryptophan fluorescence spectra of pudding, while cycles of 4/20 °C and isothermal storage at 4 °C were the most appropriate tests to accelerate changes in apparent viscosity of pudding.

High amounts of free aromatic amino acids in the protein-like fluorescence of water-dissolved organic matter

Fluorescence spectroscopy is widely used to study water pollution. The fluorescence of water natural organic matter can be classified into two groups: the protein-like fluorescence originating from aromatic amino acids and the humic fluorescence originating from humic substances. Actually, the precise molecular origin of the protein-like fluorescence is unknown because this fluorescence may be caused by either free amino acids, peptides or proteins. Therefore, we studied the molecular origin of the protein-like fluorescence of Suwannee River natural organic matter and fractions A, B and C + D obtained by size exclusion chromatography/polyacrylamide gel electrophoresis. Fractions were analyzed by reversed-phase high-performance liquid chromatography. The electrophoretic mobilities of fractions varied in the order C + D > B > A and the molecular size in the opposite order. Our results show that the protein-like fluorescence is almost exclusively located in high molecular size fraction A and medium molecular size fraction B. Retention times and fluorescence emission spectra of authentic free aromatic amino acids tyrosine and tryptophan were identical with the retention times and emission spectra of several chromatographic peaks of fractions A and B. More than 50 % of the protein-like fluorescence is due to free aromatic amino acids incorporated in water natural organic matter.

Tryptophan fluorescence quenching as a binding assay to monitor protein conformation changes in the membrane of intact mitochondria.

Intrinsic protein fluorescence is due to aromatic amino acids, mainly tryptophan, which can be selectively measured by exciting at 295nm. Changes in emission spectra of tryptophan are due to the protein conformational transitions, subunit association, ligand binding or denaturation, which affect the local environment surrounding the indole ring. In this study, tryptophan fluorescence was monitored in intact mitochondria at 333nm following excitation at 295nm in presence of insecticides using spectrofluorometer. Methyl-parathion, carbofuran, and endosulfan induced Trp fluorescence quenching and release of cytochrome c when incubated with the mitochondria, except fenvalarate. Mechanism of insecticide-induced mitochondrial toxicity for the tested insecticides has been discussed. Reduction in the intensity of tryptophan emission spectra of mitochondrial membrane proteins in presence of an increasing concentration of a ligand can be used to study the interaction of insecticides/drugs with the intact mitochondria. Furthermore, this assay can be readily adapted for studying protein-ligand interactions in intact mitochondria and in other cell organelles extending its implications for pesticide and pharma industry and in drug discovery.

Interaction of a novel antimicrobial peptide isolated from the venom of solitary bee Colletes daviesanus with phospholipid vesicles and Escherichia coli cells.

The peptide named codesane (COD), consisting of 18 amino acid residues and isolated from the venom of wild bee Colletes daviesanus (Hymenoptera : Colletidae), falls into the category of cationic α-helical amphipathic antimicrobial peptides. In our investigations, synthetic COD exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria and Candida albicans but also noticeable hemolytic activity. COD and its analogs (collectively referred to as CODs) were studied for the mechanism of their action. The interaction of CODs with liposomes led to significant leakage of calcein entrapped in bacterial membrane-mimicking large unilamellar vesicles made preferentially from anionic phospholipids while no calcein leakage was observed from zwitterionic liposomes mimicking membranes of erythrocytes. The preference of CODs for anionic phospholipids was also established by the blue shift in the tryptophan emission spectra maxima when the interactions of tryptophan-containing COD analogs with liposomes were examined. Those results were in agreement with the antimicrobial and hemolytic activities of CODs. Moreover, we found that the studied peptides permeated both the outer and inner cytoplasmic membranes of Escherichia coli. This was determined by measuring changes in the fluorescence of probe N-phenyl-1-naphthylamine and detecting cytoplasmic β-galactosidase released during the interaction of peptides with E. coli cells. Transmission electron microscopy revealed that treatment of E. coli with one of the COD analogs caused leakage of bacterial content mainly from the septal areas of the cells.

Fluorescence of tryptophan in aqueous solution

In this work, the absorption and emission spectra of Tryptophan (Trp) in aqueous solution were studied. Moreover, a hydrogen-bonded zwitterionic Trp(H2O)9 model was proposed and its ground-state and excited-state properties were investigated using the density functional theory (DFT) and the time-dependent density functional theory (TD-DFT) methods, respectively. All spectroscopic data in our experiments can be well explained by the hydrogen bond strengthening in the excited state of the model complex. The delocalization of electron density between indole moiety and neighboring H2O molecules in fluorescent state was proposed to be facilitated by the strengthened hydrogen-bond chain, and thus resulting in the large red-shift fluorescence of Trp in aqueous solution.

αs-Casein–PE6400 mixtures: a fluorescence study

Micelles are a potential encapsulation system for bioactive compounds. In previous studies we were able to show that the triblock copolymer PEO13-PPO30-PE013 and alphas-casein are miscible within distinct temperature and concentration ranges. In this study, we wanted to test our hypothesis that mixed micelles are formed which, in turn, are able to solubilize hydrophobic compounds. Additionally we want to gain insight into the specific arrangement of individual molecules in these micelles. For that purpose, mixtures containing increasing mole fractions of casein (alphacas = 4.3 x 10(-4) to alphacas = 7.2 x 10(-3)) were examined at 30 degreeC. The hydrophobic fluorescence probe pyrene was used as a model solute. Emission spectra were recorded and I1/I3 and lex/I1 ratios were evaluated. Furthermore, the emission spectra of tryptophan were recorded and the maximum emission wavelength was evaluated. The determined parameters showed that micelles are formed in all solutions and that the solubilization of pyrene occurred. The calculated interaction parameter betaindicated that the mixing was antagonistic, possibly due to steric hindrances between the casein and the polymer. It seems that the mixed micelles are formed in such a way that the hydrophobic part of the polymer is attached to the hydrophobic parts of the caseins (a necklace and bead model) since the tryptophan residues are located in a hydrophobic environment above the CMC. We suggest a first structural arrangement model, recognizing that further studies will be required to prove and refine it.

Engineering Hyperthermophilic Archaeon Pyrococcus furiosus to Overproduce Its Cytoplasmic [NiFe]-Hydrogenase

The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H(2) production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production.

Engineered tryptophan in the adenine-binding pocket of catalytic subunit A of A-ATP synthase demonstrates the importance of aromatic residues in adenine binding, forming a tool for steady-state and time-resolved fluorescence spectroscopy.

A reporter tryptophan residue was individually introduced by site-directed mutagenesis into the adenine-binding pocket of the catalytic subunit A (F427W and F508W mutants) of the motor protein A(1)A(O) ATP synthase from Pyrococcus horikoshii OT3. The crystal structures of the F427W and F508W mutant proteins were determined to 2.5 and 2.6 Å resolution, respectively. The tryptophan substitution caused the fluorescence signal to increase by 28% (F427W) and 33% (F508W), with a shift from 333 nm in the wild-type protein to 339 nm in the mutant proteins. Tryptophan emission spectra showed binding of Mg-ATP to the F427W mutant with a K(d) of 8.5 µM. In contrast, no significant binding of nucleotide could be observed for the F508W mutant. A closer inspection of the crystal structure of the F427W mutant showed that the adenine-binding pocket had widened by 0.7 Å (to 8.70 Å) in comparison to the wild-type subunit A (8.07 Å) owing to tryptophan substitution, as a result of which it was able to bind ATP. In contrast, the adenine-binding pocket had narrowed in the F508W mutant. The two mutants presented demonstrate that the exact volume of the adenine ribose binding pocket is essential for nucleotide binding and even minor narrowing makes it unfit for nucleotide binding. In addition, structural and fluorescence data confirmed the viability of the fluorescently active mutant F427W, which had ideal tryptophan spectra for future structure-based time-resolved dynamic measurements of the catalytic subunit A of the ATP-synthesizing enzyme A-ATP synthase.

Conjugates of Bovine Serum Albumin with Chitin Oligosaccharides Prepared through the Maillard Reaction

Chitin neoglycoconjugates (BSA-CO) were obtained by the conjugation of bovine serum albumin (BSA) with chitin oligosaccharides (CO) through the Maillard reaction (nonenzymatic glycation). CO produced by acid hydrolysis of chitin were fractionated using an ultrafiltration membrane system (1-3 kDa cutoff). The Maillard reaction was carried out by heating a freeze-dried mixture containing BSA and CO at 60 °C (under 43% relative humidity for 6 and 12 h). BSA-CO were characterized by available amino groups content, intrinsic tryptophan emission spectra, gel electrophoresis, and mass spectrometry. Biological assays included interaction with wheat germ agglutinin (WGA) and with bacterial adhesins of Escherichia coli K88+ and Salmonella choleraesuis. Glycation of BSA was revealed by reduction of available amino groups and fluorescence intensity and also retarded migration through SDS-PAGE. Conjugation of BSA with chitin oligomers appeared to be time dependent and was confirmed by mass spectrometry, by which molecular mass increase for monomers and dimers was observed. Monomers were estimated to contain either one or two glycation sites (at 6 and 12 h of treatment, respectively), with one or two tetrasaccharide units attached. Consequently, dimers showed two or four glycation sites. BSA-CO presented biological recognition by WGA and E. coli K88+ and S. cholerasuis adhesins. The strategy used in this work represents a simple method to obtain glycoconjugates to study applications involving protein-carbohydrate recognition.

Effect of homocysteinylation on high density lipoprotein physico-chemical properties

High density lipoproteins (HDL) exert a protective effect against homocysteinylation due to the activity of the enzyme paraoxonase/thiolactonase associated to the lipoprotein surface. However, a small amount of N-homocysteinylated HDL (N-Hcy-HDL) is present in human plasma, suggesting that homocysteinylation of plasma lipoproteins occurs in vivo. Aim of the present study was to investigate the effect of homocysteinylation on apoprotein structure and physico-chemical properties of HDL using the analysis of the fluorescent emission spectra of tryptophan and Laurdan (6-dodecanoyl-2-dimethyl-aminonaphthalene). Our results demonstrated that the increase in -SH groups in HDL homocysteinylated in vitro (Hcy-HDL) was associated with apoprotein conformational changes and modifications of physico-chemical properties. A significant decrease of paraoxonase and lactonase activity of HDL bound PON1 has also been observed in Hcy-HDL. A significant decrease of the enzyme activity has been observed also in purified PON1 homocysteinylated following the same experimental conditions used for HDL. Moreover, we demonstrated that oxidized HDL were more susceptible to homocysteinylation with respect to control HDL. The modifications of apoprotein conformation and physico-chemical properties in Hcy-HDL and the decrease of paraoxonase-1 activity could affect the protective effect of HDL against oxidative damage and/or homocysteinylation and could contribute to accelerated atherosclerosis in patients affected by diseases associated with oxidative damage, in renal disorders and in patients affected by genetic or nutritional disorders of homocysteine or folate metabolism.

Effect of phytosterols on copper lipid peroxidation of human low-density lipoproteins

Objective Phytosterols and stanols have received much attention in the past several years because of their cholesterol-lowering properties, and several studies have shown a protective effect against cardiovascular disease and colon and breast cancer development. A significant decrease of plasma low-density lipoprotein (LDL) cholesterol and apolipoprotein B has been demonstrated in subjects whose diet was supplemented with 2 g/d of plant sterols. Changes in plasma lipoprotein levels were associated with a decrease of oxidized LDL, suggesting that plant sterols could exert an antioxidant effect. The aim of the present study was to further investigate the interaction between the major dietary phytosterols and plasma lipoproteins. Moreover, their antioxidant effect against in vitro–induced lipid peroxidation of human LDL was investigated. Methods Susceptibility to copper-induced lipid peroxidation was investigated in LDLs isolated from plasma of normolipemic subjects. Concentrations of β-sitosterol, campesterol, and stigmasterol ranging from 5 to 50 μM were studied. Analyses of the emission fluorescence spectra of tryptophan and of the probe 6-dodecanoyl-2-dimethyl-aminoaphthalene were used to investigate the effect of phytosterols on apoprotein structure and physicochemical properties of LDL. Results Our results demonstrated that phytosterols exert an inhibitory effect against copper-induced lipid peroxidation of LDLs, as shown by the lowered levels of conjugated dienes in oxidized lipoproteins incubated with different concentrations of plant sterols (5–50 μM). Moreover, analysis of fluorescence emission spectra of tryptophan and 6-dodecanoyl-2-dimethyl-aminoaphthalene demonstrated that phytosterols prevent the alterations of apoprotein structure and physicochemical properties associated with copper-triggered lipid peroxidation of lipoproteins. Conclusion We suggest that the effect exerted by β-sitosterol, stigmasterol, and campesterol against lipid peroxidation of LDL possibly related to phytosterol–lipoprotein interactions could be of physiologic relevance.