Tryptophan Fluorescence Intensity Research Articles

The pH-Responsive Property of Antimicrobial Peptide GH12 Enhances Its Anticaries Effects at Acidic pH

Dental caries is closely related to the acidification of the biofilms on the tooth surface, in which cariogenic bacteria bring about a dramatic pH decrease and disrupt remineralisation equilibrium upon the fermentation of dietary sugars. Thus, approaches targeting the acidified niches with enhanced anticaries activities at acidic pH are highly desirable. In our previous study, a cationic amphipathic α-helical antimicrobial peptide GH12 (Gly-Leu-Leu-Trp-His-Leu-Leu-His-His-Leu-Leu-His-NH₂) was designed with good stability, low cytotoxicity, and excellent antibacterial effects. Considering its potent antibacterial activity against the acidogenic bacteria and its histidine-rich sequence, it was speculated that GH12 might show enhanced antimicrobial effects at an acidic pH. In this study, the pH-responsive property of GH12 was determined to evaluate its potential as a smart acid-activated anticaries agent. GH12 possessed much lower minimal inhibitory concentrations and minimal bactericidal concentrations against various kinds of bacteria at pH 5.5 than at pH 7.2. Employing Streptococcus mutans, the principal caries pathogen, as the model system, it was found that GH12 showed much stronger bactericidal effects on both planktonic S. mutans and S. mutans embedded in the biofilm at pH 5.5. In addition, short-term treatment with GH12 showed much more effective inhibitory effects on water-insoluble exopolysaccharides synthesis and lactic acid production of the preformed S. mutans biofilm at pH 5.5. As for the mechanism exploration, it was found that the net positive charge of GH12 increased and the tryptophan fluorescence intensity heightened with the peak shifting towards the short wavelength at pH 5.5, which demonstrated that GH12 could be more easily attracted to the anionic microbial cell membranes and that GH12 showed stronger interactions with the lipid membranes. In conclusion, acidic pH enhanced the antibacterial and antibiofilm activities of GH12, and GH12 is a potential smart anticaries agent targeting the cariogenic acidic microenvironment.

Effects of hydroxyl radical oxidation on myofibrillar protein and its susceptibility to μ-calpain proteolysis

In the present study, the modification of myofibrillar protein (MP) by hydroxyl radicals (·OH) and the effects of oxidative modification on the proteolysis of MP by μ-calpain were investigated. Isolated MP was exposed to different oxidation conditions and then incubated with μ-calpain. With increasing H2O2 concentration, the carbonyl and dityrosine contents as well as the surface hydrophobicity of the MP increased, while the total sulfhydryl groups and tryptophan fluorescence intensity gradually decreased. Disulfide bonds and other covalent bonds resulted in the cross-linking and aggregation of proteins. Increasing oxidant concentration resulted in the α-helix content decreasing from 19.19% to 17.32% whereas the β-sheet content increased from 21.59% to 26.47%. Protein oxidation increased the proteolytic susceptibility of actin, but inhibited the proteolysis of myosin heavy chain, troponin T, and desmin. Overall, our data demonstrate that ·OH can change the structure of MP and its susceptibility to proteolysis by μ-calpain, providing mechanistic information that links protein oxidation with proteolysis.

Co-oxidation of Antarctic krill oil with whey protein and myofibrillar protein in oil-in-water emulsions.

Antarctic krill oil (AKO) is usually encapsulated by the protein materials, enhancing its oxidative stability. Proteins exhibit immense effect on lipid oxidation and induce protein-lipid co-oxidation. This study aimed at elucidating the co-oxidation mechanism of AKO and whey protein (WP) or myofibrillar protein (MP) in oil-in-water emulsions. The estimations of malondialdehyde (MDA) content, phospholipid molecular species, and pyrrole content resulted in increased and decreased oxidation rate of AKO (especially phosphatidylethanolamine) by WP and MP, respectively. Meanwhile, protein concentration, sulfhydryl content, the loss of tryptophan fluorescence intensity, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis results demonstrated that AKO promoted WP oxidation but inhibited MP oxidation. Further, the antioxidative abilities of seven common antioxidants were evaluated. Ascorbyl palmitate showed the most substantial antioxidative effect for both AKO and proteins (about 70% decrease of MDA content and 30% decrease of the decrease ratio of tryptophan fluorescence intensity). This finding supported that different proteins could exhibit different pro/anti-oxidative effects on lipid oxidation, especially for marine lipids abundant in phospholipids and polyunsaturated fatty acids. Besides, MP could also act as antioxidant in MP AKO emulsions, further extending its application from traditional surfactants. PRACTICAL APPLICATION: AKO is usually encapsulated by the protein materials, enhancing its oxidative stability. The results demonstrated MP could inhibit AKO oxidation, and vice versa, especially when ascorbyl palmitate was added at the same time. As a result, this finding explored a new potential wall material with antioxidative ability for the encapsulated products of AKO.

In vitro digestion and cellular antioxidant activity of β-carotene-loaded emulsion stabilized by soy protein isolate-Pleurotus eryngii polysaccharide conjugates

β-Carotene is a natural fat-soluble antioxidant, while the poor oral bioavailability of β-carotene restricts its application for functional foods. To overcome this barrier, a stable oil-in-water emulsion was structured to effectively deliver β-carotene. In this study, soy protein isolate (SPI)-Pleurotus eryngii polysaccharide (PEP) conjugate was prepared by Maillard reaction under controlled wet-heating conditions. β-Carotene was encapsulated in SPI-PEP conjugate-stabilized emulsion and its gastrointestinal behavior and antioxidant activity were assessed in vitro. The results showed that such conjugate with high molecular weight was mainly formed through PEP covalently binding to lysine and cysteine of SPI via C–N. Changes in spatial structure of SPI-PEP conjugate resulted from its decreased tryptophan fluorescence intensity and unfolded protein secondary structure. A decreased surface hydrophobicity and flat surface morphology of SPI-PEP conjugate contributed to its increased water solubility and emulsibility. Additionally, lipid digestion and the formation of mixed micelles were promoted in SPI-PEP conjugate-stabilized emulsion due to its strong steric repulsion, resulting in an improved bioavailability of β-carotene. Such improvement was favorable for β-carotene to relieve tert-butyl hydroperoxide-induced oxidative stress by decreasing reactive oxygen species production and enhancing antioxidant enzyme activities in Caco-2 cells. Our study suggested that Maillard-type protein-polysaccharide conjugates-stabilized emulsions had a potential industry application for the delivery of fat-soluble nutrients.

Comparison of the in vitro protein digestibility of Protaetia brevitarsis larvae and beef loin before and after defatting

Protein digestibility of Protaetia brevitarsis larvae before and after defatting by hexane was compared with that of beef loin in an in vitro digestion model. Larvae had higher crude protein content and 10% trichloroacetic acid (10% TCA)-soluble α-amino groups than beef. Decreases in the levels of total free sulfhydryl groups and 10% TCA-soluble α-amino groups were detected in larvae and beef after defatting (P < 0.05). Surface hydrophobicity increased after defatting in both larvae and beef, (P < 0.05) and tryptophan fluorescence intensity decreased in defatted larvae but increased in defatted beef. Levels of proteins digested into sizes under 3 and 10 kDa in larvae were higher than those in beef (P < 0.05), and defatting did not induce an effect in larvae. Therefore, in the aspect of high protein content and digestibility, larvae of P. brevitarsis can be a potential substitute of animal proteins.

Changes in the Functional Activity of Horseradish Peroxidase and Bovine Serum Albumin in Media with Different Isotope 2H/1H Compositions

Abstract—It is established that a medium with a reduced content of deuterium has no effect on the secondary structure of horseradish peroxidase and bovine serum albumin and caused no conformational changes in the structures of these proteins. The placement of these proteins in a buffer solution prepared based on deuterium-depleted water led to a decrease in the intensity of intrinsic tryptophan fluorescence, while the circular dichroism spectra remained virtually unchanged. A decrease in the content of deuterium in the reaction medium led to a decrease in the activity of the peroxidase oxidation reaction of o-dianisidine and luminal with hydrogen peroxide.

Fluorescence-based monitoring of anthropogenic pollutant inputs to an urban stream in Southern California, USA

Fluorescence spectroscopy has been increasingly used to detect sewage and other anthropogenic contaminants in surface waters. Despite progress in successfully detecting bacterial and sewage inputs to rivers over diverse spatial scales, the use of fluorescence-based in-situ sensors to track contaminant inputs during storm events and to discern bacterial contamination from background natural organic matter (NOM) fluorescence have received less attention. A portable, submersible fluorometer equipped with tryptophan (TRP)-like and humic-like fluorescence sensors was used to track inputs of untreated wastewater added to natural creek water in a laboratory sewage spill simulation. Significant, positive correlations were observed between TRP fluorescence, the TRP:humic ratio, percent wastewater, and Escherichia coli concentrations, indicating that both the TRP sensor and the TRP:humic ratio tracked wastewater inputs against the background creek water DOM fluorescence. The portable fluorometer was subsequently deployed in an urban creek during a storm in 2018. The peak in TRP fluorescence was found to increase with the rising limb of the hydrograph and followed similar temporal dynamics to that of caffeine and fecal indicator bacteria, which are chemical and biological markers of potential fecal pollution. Results from this study demonstrate that tracking of TRP fluorescence intensity and TRP:humic ratios, with turbidity correction of sensor outputs, may be an appropriate warning tool for rapid monitoring of sewage or other bacterial inputs to aquatic environments.

Unraveling the Origin of Multi-Exponential Fluorescence Intensity Decay of Tryptophan in Proteins

The multi-exponential nature of tryptophan fluorescence intensity decay among proteins has puzzled biophysicists over all these years. Its origin has been previously attributed to, emission from multiple electronic absorption bands, namely La and Lb states; presence of a mixture of Trp rotamers that possess unique fluorescence lifetimes or multiple conformational states of the protein. However, none of the above explanations have been proved conclusive. Here, we report that the multi-exponential decay originates due to additional luminescence contribution from protein charge transfer states. We have previously shown (Chem. Sci., 2017, 8, 5416) the existence of weak but significant electronic absorption between 250—800 nm among proteins rich in charged residues resulting in Protein Charge Transfer Spectra (ProCharTS). Separated charges in the protein excited state created by photoinduced electron transfer emit weak luminescence on charge recombination. We observed that fluorescence intensity decay of N-Acetyl-L-Tryptophanamide (NATA) on excitation at 295 nm showed gradual deviation from mono-exponential decay kinetics with increasing presence of different charged amino acids (D, E, K, H and R). Notably contribution from a ∼2 ns component showed a significant increase in presence of charged amino acids at expense of ∼3 ns component arising from indole. The presence of charged amino acids also quenched its fluorescence lifetime (from ∼3 to ∼ 2.3 ns). Further, influence of ProCharTS was not restricted to intrinsic fluorophore like Trp. A charge rich protein like human serum albumin on conjugation with Dansyl (2-dimethyl amino naphthalene 6-sulphonyl chloride) probe displays a low mean fluorescence lifetime of ∼6.5 ns, while the same probe on conjugation to relatively less charge rich protein like hen lysozyme displays a lifetime of ∼14 ns. Thus, ProCharTS emission has a significant role in origin of multi‒exponential decay kinetics of Trp in proteins.

Site-specific incorporation of sodium tripolyphosphate into myofibrillar protein from mantis shrimp (Oratosquilla oratoria) promotes protein crosslinking and gel network formation

Formation of protein gels in processed muscle foods is one of the most important functionalities. To explore the mechanisms responsible for affecting gel properties of muscle proteins by phosphates, myofibrillar protein (MP) from mantis shrimp (Oratosquilla oratoria) was treated with sodium tripolyphosphate at three pH values (7.0, 8.0, and 9.0). FTIR and UPLC-MS/MS firstly confirmed that phosphate groups were introduced to MP through COP bonds via serine and threonine residues. The incorporation of STP caused increased electronegativity and solubility, more stable α-helix secondary conformation, and reduced tryptophan fluorescence intensity of MP, especially at pH 8.0 and 9.0. These changes led to a finer, ordered and denser three-dimensional gel network microstructure with higher gel strength and elasticity, and water-holding capacity. This study demonstrated that the introduction of phosphate groups could increase negatively charged residues in MP, enhance the crosslinks of proteins through ionic interaction, and promote gel network formation.

Effect of mutation at positively charged residues (K329 and R330) in a flexible region of firefly luciferase on structure and kinetic properties

Firefly luciferase as a bioluminescent enzyme has many applications in various fields from scientific research to commercial goals. This enzyme is relatively unstable with low functional capacity due to rapid inactivation in physiological temperature, low in vitro stability and high susceptibility to proteolytic degradation. Based on previous studies, two regions 206–220 and 329–341 on N-domain of Photinus pyralis luciferase are known accessible and flexible. Flexible regions may lead to protein instability. Here, the effect of mutation at positively charged residues Lys(K)329 and Arg(R)330 on the stability of luciferase was studied. Furthermore, the role of these mutations on the structure and function was evaluated. Introducing of these point mutations did not affect the orientation of critical residues in bioluminescence color determination. The kinetic studies showed that thermostability and Km value for luciferin in both mutants were decreased as compared to wild type. However, optimum pH and optimum temperature showed no significant changes in both mutants. Moreover, the structural data revealed an increase in tryptophan fluorescence intensity and secondary structure content for R330Q in compared with wild type, while intrinsic fluorescence and far-UV CD intensity in K329I mutant was decreased.

Combined effect of ultrasound and sodium bicarbonate marination on chicken breast tenderness and its molecular mechanism

The objective of this study was to explore the combined effect of ultrasound (20 kHz ultrasound probe (12 mm diameter), an output power of 350 W for 5 min (on-time and off-time pulse durations of 2 s and 3 s, respectively)) with low concentration (0.2 M) sodium bicarbonate solution (USB) on chicken breast tenderness and its preliminary molecular mechanism. Therefore, myofibril fragmentation index (MFI), filtering residues, cooking loss, shear force, histology of meat as well as SDS-PAGE, circular dichroism (CD), synchronous fluorescence spectroscopy, differential scanning calorimetrv (DSC) of actomyosin were investigated and compared with untreated (Control), deionized water (DW), ultrasound in deionized water (UDW), 0.2 M sodium bicarbonate solution (SB), respectively. The results showed that USB can effectively increase MFI, and reduce filtering residues, cooking loss and shear force by compared with UDW or SB. Furthermore, myofibril was presented with the largest interfibrillary spaces and the highest degree of actomyosin dissociation in USB group. The increase in α-helix content and decrease in fluorescence intensity of tyrosine and tryptophan implied that USB caused the conformation change in actomyosin. Additionally, actomyosin in USB group became more sensitive to temperature. Therefore, the treatment of ultrasound combined with low concentration of sodium bicarbonate accelerated actomyosin degeneration was considered as a promising and efficient technique in meat processing, especially for the fitness enthusiasts.

Tetramethylpyrazine Protects Oxidative Stability and Gelation Property of Rabbit Myofibrillar Proteins.

Tetramethylpyrazine (TMP), an alkaloid rich in Ligusticum wallichii and fermented products, possesses multiple pharmacological activities in antioxidant, anti-inflammatory, and antibacterial. This study aimed to investigate the effect of TMP (15 mg/L) on the physicochemical and gelation properties of rabbit myofibrillar proteins (MPs) with/without oxidative stress. Results showed that compared to the control, oxidative stress to MPs decreased free thiol content, gel yield, whiteness, water-holding capacity, bounder water, immobilized water, and endogenous tryptophan fluorescence intensity, but increased surface hydrophobicity, dityrosine content, and free water content (p<0.01). Without oxidative stress, MPs treated with TMP increased free thiol content, whiteness, and bound water, but decreased dityrosine content and free water (p<0.05). Under oxidative conditions, all parameters were conversely affected by TMP (p<0.01). The results suggest that TMP can be an antioxidant to decrease the concern on oxidative deterioration during meat processing and storage by improving the oxidative stability, water retention, and gel forming property of rabbit MPs.

Genetic variation and potential for genetic improvement of cuticle deposition on chicken eggs

BackgroundThe cuticle is an invisible glycosylated protein layer that covers the outside of the eggshell and forms a barrier to the transmission of microorganisms. Cuticle-specific staining and in situ absorbance measurements have been used to quantify cuticle deposition in several pure breeds of chicken. For brown eggs, a pre-stain and a post-stain absorbance measurement is required to correct for intrinsic absorption by the natural pigment. For white eggs, a post-stain absorbance measurement alone is sufficient to estimate cuticle deposition. The objective of the research was to estimate genetic parameters and provide data to promote adoption of the technique to increase cuticle deposition and reduce vertical transmission of microorganisms.ResultsFor all pure breeds examined here, i.e. Rhode Island Red, two White Leghorns, White Rock and a broiler breed, the estimate of heritability for cuticle deposition from a meta-analysis was moderately high (0.38 ± 0.04). In the Rhode Island Red breed, the estimate of the genetic correlation between measurements recorded at early and late times during the egg-laying period was ~ 1. There was no negative genetic correlation between cuticle deposition and production traits. Estimates of the genetic correlation of cuticle deposition with shell color ranged from negative values or 0 in brown-egg layers to positive values in white- or tinted-egg layers. Using the intrinsic fluorescence of tryptophan in the cuticle proteins to quantify the amount of cuticle deposition failed because of complex quenching processes. Tryptophan fluorescence intensity at 330 nm was moderately heritable, but there was no evidence of a non-zero genetic correlation with cuticle deposition. This was complicated furthermore by a negative genetic correlation of fluorescence with color in brown eggs, due to the quenching of tryptophan fluorescence by energy transfer to protoporphyrin pigment. We also confirmed that removal of the cuticle increased reflection of ultraviolet wavelengths from the egg.ConclusionsThese results provide additional evidence for the need to incorporate cuticle deposition into breeding programs of egg- and meat-type birds in order to reduce vertical and horizontal transmission of potentially pathogenic organisms and to help improve biosecurity in poultry.

Extracting the protein dynamics of bovine serum albumin in the native condition using confocal fluorescent temperature jump

A second-resolved temperature jump (T-jump) system coupled with confocal fluorescent thermometry of 100-μm spatial resolution was employed to unravel the protein dynamics of bovine serum albumin (BSA) at initial temperatures of 25–42 °C. The different evolutions of the tryptophan fluorescence intensity change of the BSA and pure tryptophan upon increasing 5 °C from 25 °C indicated an intrinsic protein dynamics quantified with an apparent rise time of 27 ± 1 s, whereas such a process was not observed at initial temperatures >30 °C. Different heating rates could differentiate the reaction pathways to detour toward reactions possessing different apparent activation energies, successfully explaining the discrepancy of the fluorescence intensity change observed in steady-state and T-jump methods. Moreover, the temperature-dependent steady-state circular dichroism and tryptophan fluorescence spectra were not coincident because they reflected the global and localized structural alterations upon heating, respectively. The combination of the experimental approach and concomitant analysis of the fluorescence evolutions provides a new tool to illustrate the dynamic processes of a big protein upon thermal stimulus.

Protective effects of epicatechin on the oxidation and N-nitrosamine formation of oxidatively stressed myofibrillar protein

ABSTRACT The present study investigated the protective effects of the polyphenol epicatechin on the protein structural changes, gel properties and N-nitrosodiethylamine (NDEA) formation of oxidatively stressed myofibrillar protein (MP). Results showed that oxidation treatments to MP significantly increased their carbonyl content and surface hydrophobicity as compared to the control groups (p < 0.05). Epicatechin inhibited protein carbonyl content and surface hydrophobicity, especially treated with 2 mM epicatechin. Under oxidative conditions, the fluorescence intensity of tryptophan gradually decreased with the increase of epicatechin concentrations from 0 to 2 mM. The storage modulus (G’) was increased gradually after oxidation and epicatechin treatment. Epicatechin reduced the loss of P 22 and water holding capacity (WHC), promoted the hydration of the proteins. Under oxidative conditions, epicatechin had a concentration dependent effect on the formation of NDEA; low concentrations of epicatechin promoted NDEA formation, while high concentrations inhibited NDEA production. In conclusion, an appropriate quantity of polyphenols can inhibit the oxidation of MP and formation of N-nitrosamines.

Acidulant effect on greening, reducing capacity, and tryptophan fluorescence of sunflower butter cookie dough during refrigerated storage.

Sunflower seed derived butter can be a source of protein and phenolic antioxidants in refrigerated dough. Chlorogenic quinone-amino acid induced greening can however occur at alkaline pH, which could result in less bioavailable conjugated phenol-amino acids. Acidulants were tested as potential anti-greening ingredients in refrigerated chemically leavened cookie dough. Effect of refrigerated storage time, leavening agents and acidulants on tryptophan fluorescence (λex=280 nm, λem=300-500 nm), color (hunter L*, a*, b* color scale), reducing capacity [1,1'-diphenyl-2-picryl-hydrazyl (DPPH) and Folin-Ciocalteu reducing capacity (FCRC)], and hydroxycinnamic acids were measured. The pH range of acidified doughs was 4.83-6.98 compared to 7.65-9.18 in non-acidified leavened doughs after 24 days. Greening was higher in baking soda dough control (a*=-0.54) than baking powder dough control (a*=2.98) after 24 days, attributed to higher pH (9.18) of the former compared to pH7.14 in the later. Tryptophan fluorescence intensity in baking soda dough decreased in the order: control>glucono-delta lactone≈citric acid after 24 days. The DPPH and FCRC of acidified doughs were greater than corresponding control doughs. The use of acidulants would prevent greening in sunflower dough without lowering its phenolic concentration, making use of sunflower butter in refrigerated dough for baked goods feasible. © 2018 Society of Chemical Industry.

The antimicrobial spectrum of lysozyme broadened by reductive modification.

In order to broaden the antimicrobial action spectrum of lysozyme against Gram-negative bacteria, different partially reduced lysozyme derivatives were obtained by reducing the R-S-S-R bonds of lysozyme using the reducing agent Na2SO3. The circular dichroism behavior analysis showed that the tertiary structure of the partially reduced lysozyme molecules became more flexible, leading to an obvious increase in the surface hydrophobicity and the tryptophan fluorescence intensity. Zeta potential analysis indicated that the introduction of SO32- led to a decrease in the surface charges of the reduced lysozyme molecules. Compared with the native lysozyme, the reduced lysozymes not only showed a 1 log increase in the antimicrobial activity against Escherichia coli ATCC 25922 and Salmonella enteritidis ATCC 13076 (P <0.05), but also maintained nearly the same antimicrobial activity against Staphylococcus aureus ATCC 29213 (P >0.05). Overall, the reductive modification with the food-friendly, compatible, and safe reducing agent Na2SO3 has broadened the antimicrobial action spectrum of the modified lysozymes against Gram-negative bacteria, with the enhancement extent depending on the reduction degree and the type of bacterial strains. The integrated results suggest that the Na2SO3-reduced lysozyme can be used as a novel safe potential bactericidal additive for food-processing industry.

Sulfur dioxide induced aggregation of wine thaumatin-like proteins: Role of disulfide bonds

Aggregation of heat unstable wine proteins is responsible for the economically and technologically detrimental problem called wine protein haze. This is caused by the aggregation of thermally unfolded proteins that can precipitate in bottled wine. To study the influence of SO2 in this phenomenon, wine proteins were isolated and thaumatins were identified has the most prone to aggregate in the presence of this compound. Isolated wine thaumatins aggregation was followed by dynamic light scattering (DLS), circular dichroism (CD), fluorescence spectroscopy and size exclusion chromatography (SEC). Our experimental results demonstrate that protein thermal unfolding after exposure of the protein to 70 °C does not present differences whether SO2 is present or not. Conversely, when the protein solution is cooled to 15 °C (after heat stress) significant analytical changes can be observed between samples with and without SO2. A remarkable change of circular dichroism spectra in the region 220–230 nm is observed (which can be related to S-S torsion angles), as well as an increase in tryptophan fluorescence intensity (absence of fluorescence quenching by S-S bonds). Formation of covalently-linked dimeric and tetrameric protein species were also detected by SEC. The ability to dissolve the aggregates with 8 M urea seems to indicate that hydrophobic interactions are prevalent in the formed aggregates. Also, the reduction of these aggregates with tris (2-carboxyethyl) phosphine (TCEP) to only monomeric species reveals the presence of intermolecular S-S bonds.

Calorimetric and Spectroscopic Studies of the Effects of the Cell Penetrating Peptide Pep-1 and the Antimicrobial Peptide Combi-2 on Vesicles Mimicking Escherichia coli Membrane.

The objective of this study is to measure and compare the effects of the cell penetrating peptide (CPP) Pep-1 and the antimicrobial peptide (AMP) combi-2 on vesicles of membranes mimicking Escherichia coli (E. coli). To characterize the effects of Pep-1 and combi-2 on E. coli membrane vesicles, a combination of five biophysical techniques was employed: fluorescence, infrared, scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC) techniques. Upon addition of E. coli membranes, tryptophan fluorescence intensity of Pep-1 showed a sudden blue-shift and decreased in a nonconcentration-dependent manner while the intensity of combi-2 decreased in a concentration-dependent manner, most significantly for a very low peptide-to-lipid ratio of 1:40. Complexes of Pep-1 and combi-2 with E. coli membrane mimicking vesicles having shown a significant blue-shift in fluorescence intensity were then prepared and studied in freeze-dried states. IR results indicate that Pep-1 and combi-2 adopt a major 310-helix structure in the presence of E. coli membrane mimicking vesicles at low peptide concentration. Pep-1 and combi-2 have a similar effect on E. coli membrane mimicking vesicles at low concentration even though combi-2 is in the interfacial region of the bilayer while Pep-1 is located between the interfacial region and the hydrophobic region. Combi-2 at low concentration acts as a CPP. TGA and DSC results reveal that combi-2 has a stabilizing effect on E. coli at any concentration while Pep-1 stabilizes the E. coli membrane only at high concentration. Both peptides show a preferential interaction with one of the anionic lipids leading to clustering in E. coli membrane. SEM images reveal that Pep-1 and combi-2 form superstructures including fibrils in the presence of E. coli membrane mimicking vesicles. Calorimetric and spectroscopic techniques may be used in a complementary way with imaging techniques to gain more insights into peptide-lipid interactions.

Spectroscopic Study of the Interactions of Ruthenium-Ketoconazole Complexes of Known Antiparasitic Activity with Human Serum Albumin and Apotransferrin

The pharmacological properties of any drug are related to their ability to interact with macromolecular blood components. The interaction of human serum albumin (HSA) and apotransferrin (ATf) with six RuII complexes containing ketoconazole (KTZ), which we have previously reported to be active against Leishmania major and Trypanosoma cruzi, has been investigated by monitoring the tryptophan fluorescence intensity of each protein upon incremental addition of the complexes. All the Ru-KTZ derivatives, namely cis-fac-[RuIICl2(DMSO)3(KTZ)] (1), cis-[RuIICl2(bipy)(DMSO)(KTZ)] (2), [RuII(η6-p-cymene)Cl2(KTZ)] (3), [RuII(η6-p-cymene)(en)(KTZ)][BF4]2 (4), [RuII(η6-p-cymene)(bipy)(KTZ)][BF4]2 (5), and [RuII(η6-p-cymene)(acac)(KTZ)][BF4] (6) are able to quench the intrinsic fluorescence of HSA and ATf at 27 °C. Analysis of the spectroscopic data using Stern-Volmer plots indicates that in both cases the quenching takes place principally through a static mechanism involving the formation of Ru complex-protein adducts; further analysis of the fluorescence data allowed the estimation of apparent association constants and the number of binding sites for each protein and each compound. The results indicate that both HSA and ATf are possible effective transporters for Ru-KTZ antiparasitic drugs.