Seasonal studies of aquatic humic substances from Amazon rivers: characterization and interaction with Cu (II), Fe (II), and Al (III) using EEM-PARAFAC and 2D FTIR correlation analyses.

Aquatic humic substances (AHS) are defined as an important components of organic matter, being composed as small molecules in a supramolecular structure and can interact with metallic ions, thereby altering the bioavailability of these species. To better understand this behavior, AHS were extracted and characterized from Negro River, located near Manaus city and Carú River, that is situated in Itacoatiara city, an area experiencing increasing anthropogenic actions; both were characterized as blackwater rivers. The AHS were characterized by 13C nuclear magnetic ressonance and thermochemolysis GC-MS to obtain structural characteristics. Interaction studies with Cu (II), Al (III), and Fe (III) were investigated using fluorescence spectroscopy applied to parallel factor analysis (PARAFAC) and two-dimensional correlation spectroscopy with Fourier transform infrared spectroscopy (2D-COS FTIR). The AHS from dry season had more aromatic fractions not derived from lignin and had higher content of alkyls moities from microbial sources and vegetal tissues of autochthonous origin, while AHS isolated in the rainy season showed more metals in its molecular architecture, lignin units, and polysacharide structures. The study showed that AHS composition from rainy season were able to interact with Al (III), Fe (III), and Cu (II). Two fluorescent components were identified as responsible for interaction: C1 (blue-shifted) and C2 (red-shifted). C1 showed higher complexation capacities but with lower complexation stability constants (KML ranged from 0.3 to 7.9 × 105) than C2 (KML ranged from 3.1 to 10.0 × 105). 2D-COS FTIR showed that the COO- and C-O in phenolic were the most important functional groups for interaction with studied metallic ions.

A Computational-Experimental Investigation of the Molecular Mechanism of Interleukin-6-Piperine Interaction.

Herein, we elucidate the biophysical aspects of the interaction of an important protein, Interleukin-6 (IL6), which is involved in cytokine storm syndrome, with a natural product with anti-inflammatory activity, piperine. Despite the role of piperine in the inhibition of the transcriptional protein NF-κB pathway responsible for activation of IL6 gene expression, there are no studies to the best of our knowledge regarding the characterisation of the molecular interaction of the IL6-piperine complex. In this context, the characterisation was performed with spectroscopic experiments aided by molecular modelling. Fluorescence spectroscopy alongside van't Hoff analyses showed that the complexation event is a spontaneous process driven by non-specific interactions. Circular dichroism aided by molecular dynamics revealed that piperine caused local α-helix reduction. Molecular docking and molecular dynamics disclosed the microenvironment of interaction as non-polar amino acid residues. Although piperine has three available hydrogen bond acceptors, only one hydrogen-bond was formed during our simulation experiments, reinforcing the major role of non-specific interactions that we observed experimentally. Root mean square deviation (RMSD) and hydrodynamic radii revealed that the IL6-piperine complex was stable during 800 ns of simulation. Taken together, these results can support ongoing IL6 drug discovery efforts.

Experimental Approaches and Computational Modeling of Rat Serum Albumin and Its Interaction with Piperine.

The bioactive piperine (1-piperoyl piperidine) compound found in some pepper species (Piper nigrum linn and Piper sarmentosum Roxb) has been shown to have therapeutic properties and to be useful for well-being. The tests used to validate these properties were performed in vitro or with small rats. However, in all these assays, the molecular approach was absent. Although the first therapeutic trials relied on the use of rats, no proposal was mentioned either experimentally or computationally at the molecular level regarding the interaction between piperine and rat serum albumin (RSA). In the present study, several spectroscopic techniques were employed to characterize rat serum albumin and, aided by computational techniques, the protein modeling was proposed. From the spectroscopic results, it was possible to estimate the binding constant (3.9 × 104 M-1 at 288 K) using the Stern-Volmer model and the number of ligands (three) associated with the protein applying interaction density function model. The Gibbs free energy, an important thermodynamic parameter, was determined (-25 kJ/mol), indicating that the interaction was spontaneous. This important set of experimental results served to parameterize the computational simulations. The results of molecular docking and molecular dynamics matched appropriately made it possible to have detailed microenvironments of RSA accessed by piperine.

Annexin A1 Mimetic Peptide and Piperlongumine: Anti-Inflammatory Profiles in Endotoxin-Induced Uveitis.

Uveitis is one of the main causes of blindness worldwide, and therapeutic alternatives are worthy of study. We investigated the effects of piperlongumine (PL) and/or annexin A1 (AnxA1) mimetic peptide Ac2-26 on endotoxin-induced uveitis (EIU). Rats were inoculated with lipopolysaccharide (LPS) and intraperitoneally treated with Ac2-26 (200 µg), PL (200 and 400 µg), or Ac2-26 + PL after 15 min. Then, 24 h after LPS inoculation, leukocytes in aqueous humor, mononuclear cells, AnxA1, formyl peptide receptor (fpr)1, fpr2, and cyclooxygenase (COX)-2 were evaluated in the ocular tissues, along with inflammatory mediators in the blood and macerated supernatant. Decreased leukocyte influx, levels of inflammatory mediators, and COX-2 expression confirmed the anti-inflammatory actions of the peptide and pointed to the protective effects of PL at higher dosage. However, when PL and Ac2-26 were administered in combination, the inflammatory potential was lost. AnxA1 expression was elevated among groups treated with PL or Ac2-26 + PL but reduced after treatment with Ac2-26. Fpr2 expression was increased only in untreated EIU and Ac2-26 groups. The interaction between Ac2-26 and PL negatively affected the anti-inflammatory action of Ac2-26 or PL. We emphasize that the anti-inflammatory effects of PL can be used as a therapeutic strategy to protect against uveitis.

The Specific Elongation Factor to Selenocysteine Incorporation in Escherichia coli: Unique tRNASec Recognition and its Interactions.

Several molecular mechanisms are involved in the genetic code interpretation during translation, as codon degeneration for the incorporation of rare amino acids. One mechanism that stands out is selenocysteine (Sec), which requires a specific biosynthesis and incorporation pathway. In Bacteria, the Sec biosynthesis pathway has unique features compared with the eukaryote pathway as Ser to Sec conversion mechanism is accomplished by a homodecameric enzyme (selenocysteine synthase, SelA) followed by the action of an elongation factor (SelB) responsible for delivering the mature Sec-tRNASec into the ribosome by the interaction with the Selenocysteine Insertion Sequence (SECIS). Besides this mechanism being already described, the sequential events for Sec-tRNASec and SECIS specific recognition remain unclear. In this study, we determined the order of events of the interactions between the proteins and RNAs involved in Sec incorporation. Dissociation constants between SelB and the native as well as unacylated-tRNASec variants demonstrated that the acceptor stem and variable arm are essential for SelB recognition. Moreover, our data support the sequence of molecular events where GTP-activated SelB strongly interacts with SelA.tRNASec. Subsequently, SelB.GTP.tRNASec recognizes the mRNA SECIS to deliver the tRNASec to the ribosome. SelB in complex with its specific RNAs were examined using Hydrogen/Deuterium exchange mapping that allowed the determination of the molecular envelopes and its secondary structural variations during the complex assembly. Our results demonstrate the ordering of events in Sec incorporation and contribute to the full comprehension of the tRNASec role in the Sec amino acid biosynthesis, as well as extending the knowledge of synthetic biology and the expansion of the genetic code.

Fluorescence spectroscopic and dynamics simulation studies on isoorientin binding with human serum albumin.

Isoorientin (ISOO) a glycosylated flavonoid found in acai berry exhibits relevant activities such as antidiabetic and antidepressant. However, its physicochemical action on any molecular target is scarcely known. In this work, we tackle the problem about the binding of ISOO to human serum albumin (HSA) applying fluorescence spectroscopy bimodal analysis aided by computational simulations. A static quenching process was detected having hypsochromic shift with implication in the polarizability around the endogenous probe (Trp 214) during complex formation. The binding mechanism reveals that all sites are equivalents and independents with binding constant value of 9.1 × 104 M-1 and, a total of six sites accessed whereas three of them were identified experimentally. The thermodynamic evaluation indicates that the complex formation is spontaneous (ΔG<0). The dynamics and docking simulations corroborated the experimental data by adding details of each site and its respective microenvironment.

Detailed Characterization of the Cooperative Binding of Piperine with Heat Shock Protein 70 by Molecular Biophysical Approaches.

In this work, for the first time, details of the complex formed by heat shock protein 70 (HSP70) independent nucleotide binding domain (NBD) and piperine were characterized through experimental and computational molecular biophysical methods. Fluorescence spectroscopy results revealed positive cooperativity between the two binding sites. Circular dichroism identified secondary conformational changes. Molecular dynamics along with molecular mechanics Poisson Boltzmann surface area (MM/PBSA) reinforced the positive cooperativity, showing that the affinity of piperine for NBD increased when piperine occupied both binding sites instead of one. The spontaneity of the complexation was demonstrated through the Gibbs free energy (∆G < 0 kJ/mol) for different temperatures obtained experimentally by van't Hoff analysis and computationally by umbrella sampling with the potential of mean force profile. Furthermore, the mean forces which drove the complexation were disclosed by van't Hoff and MM/PBSA as being the non-specific interactions. In conclusion, the work revealed characteristics of NBD and piperine interaction, which may support further drug discover studies.

The Cytokine IL-1ß and Piperine Complex Surveyed by Experimental and Computational Molecular Biophysics.

The bioactive piperine, a compound found in some pepper species, has been widely studied because of its therapeutic properties that include the inhibition of an important inflammation pathway triggered by interleukin-1 beta (IL-1ß). However, investigation into the molecular interactions between IL-1ß and piperine is not reported in the literature. Here, we present for the first time the characterisation of the complex formed by IL-1ß and piperine through experimental and computational molecular biophysical analyses. Fluorescence spectroscopy unveiled the presence of one binding site for piperine with an affinity constant of 14.3 × 104 M-1 at 298 K. The thermodynamic analysis indicated that the interaction with IL-1ß was spontaneous (∆G = -25 kJ/mol) and, when split into enthalpic and entropic contributions, the latter was more significant. Circular dichroism spectroscopy showed that piperine did not affect IL-1ß secondary structure (~2%) and therefore its stability. The set of experimental data parameterized the computational biophysical approach. Through molecular docking, the binding site micro-environment was revealed to be composed mostly by non-polar amino acids. Furthermore, molecular dynamics, along with umbrella sampling, are in agreement with the thermodynamic parameters obtained by fluorescence assays and showed that large protein movements are not present in IL-1ß, corroborating the circular dichroism data.

Humic extracts from hydrochar and Amazonian Anthrosol: Molecular features and metal binding properties using EEM-PARAFAC and 2D FTIR correlation analyses.

Organic matter plays many roles in the soil ecosystem. One property of the substance concerns the metal complexation and interaction with organic contaminants. In this sense, the humic substances (HS), a heterogeneous mixture of compounds, naturally derived from degradation of biomass, have been widely studied in environmental sciences. Recent advances showed a new way to produce humic-like substances (HLS) through hydrothermal carbonization of biomass. Thus, this study aimed to evaluate the HLS of hydrochars, produced by using a mixture of sugarcane bagasse and vinasse with sulfuric acid added (1 and 4% v/v), and to assess their interactions with metal ions, (Fe(III), Al(III), Cu(II) and Co(II)) using EEM-PARAFAC and a two-dimensional FTIR correlation analysis. The results were compared to the humic substances extracted from the Amazonian Anthrosol, as a model of anthropogenic organic matter. NMR analysis showed that humic-like extracts from hydrochar are mainly hydrophobic, while the soil has a greater contribution of polar moieties. The HLS and HS showed similar complexation capacities for Fe(III), Al(III) and Cu(II) assays. For Co(II) HLS exhibited larger affinities than HS. Two-dimensional correlation analysis FTIR showed that chemical groups may undergo conformational alteration with metal additions to achieve more stable arrangements (higher stability constant). Therefore, these results contribute more knowledge about the mechanism of HS and metal ion interaction, as well as showing that HTC can be an interesting option for HLS production, to be used as humic based materials.

Details of the cooperative binding of piperlongumine with rat serum albumin obtained by spectroscopic and computational analyses.

Piperlongumine (PPL) has presented a variety of important pharmacological activities. In recent pharmacokinetics studies in rats, this molecule reached 76.39% of bioavailability. Although PPL is present in the bloodstream, no information is found on the interaction between PPL and rat serum albumin (RSA), the most abundant protein with the function of transporting endo/exogenous molecules. In this sense, the present study elucidated the mechanism of interaction between PPL and RSA, using in conjunction spectroscopic and computational techniques. This paper shows the importance of applying inner filter correction over the entire fluorescence spectrum prior to any conclusion regarding changes in the polarity of the fluorophore microenvironment, also demonstrates the convergence of the results obtained from the treatment of fluorescence data using the area below the spectrum curve and the intensity in a single wavelength. Thermodynamic parameters revealed that PPL binds to RSA spontaneously (ΔG < 0) and the process is entropically driven. Interaction density function method (IDF) indicated that PPL accessed two cooperative sites in RSA, with moderate binding constants (2.3 × 105 M-1 and 1.3 × 105 M-1). The molecular docking described the microenvironment of the interaction sites, rich in apolar residues. The stability of the RSA-PPL complex was checked by molecular dynamics.

An investigation into the interaction between piplartine (piperlongumine) and human serum albumin.

Piplartines are alkaloid amides present in the roots and stems of different pepper species which have promising pharmacological properties including cancer prevention. Some recent studies have determined pharmacokinetic parameters of piplartine in rat blood plasma but without pointing to any molecular target or describing the physicochemical forces of the interaction. The present study investigated the interaction between piplartine and human serum albumin (HSA) the predominant protein in blood plasma. Fluorescence spectroscopy was utilized to observe the complex HSA-piplartine formation. Thermodynamic parameter analysis indicates that the process occurs spontaneously and is enthalpically driven; the affinity constant suggests that this interaction is reversible. This was reinforced by the binding density function method and by the displacement analysis that the piplartine binds on HSA at a single site, which was determined to be the IIA sub-domain. In silico analysis (molecular docking) identified the main residues involved in binding and the corresponding forces, which corroborates well with the experimental results.

Spectral and computational features of the binding between riparins and human serum albumin.

The green Brazilian bay leaf, a spice much prized in local cuisine (Aniba riparia, Lauraceae), contains chemical compounds presenting benzoyl-derivatives named riparins, which have anti-inflammatory, antimicrobial and anxiolytic properties. However, it is unclear what kind of interaction riparins perform with any molecular target. As a profitable target, human serum albumin (HSA) is one of the principal extracellular proteins, with an exceptional capacity to interact with several molecules, and it also plays a crucial role in the transport, distribution, and metabolism of a wide variety of endogenous and exogenous ligands. To outline the HSA-riparin interaction mechanism, spectroscopy and computational methods were synergistically applied. An evaluation through fluorescence spectroscopy showed that the emission, attributed to Trp 214, at 346 nm decreased with titrations of riparins. A static quenching mechanism was observed in the binding of riparins to HSA. Fluorescence experiments performed at 298, 308 and 318 K made it possible to conduct thermodynamic analysis indicating a spontaneous reaction in the complex formation (ΔG<0). The enthalpy-entropy balance experiment with a molecular modeling calculation revealed that hydrophobic, hydrogen bond and non-specific interactions are present for riparin I-III with HSA. The set of results from fractional fluorescence changes obtained through Schatchard was inconclusive in establishing what kind of cooperativity is present in the interaction. To shed light upon the HSA-riparins complex, Hill's approach was utilized to distinguish the index of affinity and the binding constant. A correspondence between the molecular structures of riparins, due to the presence of the hydroxyl group in the B-ring, with thermodynamic parameters and index of affinity were observed. Riparin III performs an intramolecular hydrogen bond, which affects the Hill coefficient and the binding constant. Therefore, the presence of hydroxyl groups is capable of modulating the interaction between riparins and HSA. Site marker competitive experiments indicated Site I as being the most suitable, and the molecular modeling tools reinforced the experimental results detailing the participation of residues.

An integrated approach with experimental and computational tools outlining the cooperative binding between 2-phenylchromone and human serum albumin.

2-Phenylchromone (2PHE) is a flavone, found in cereals and herbs, indispensable in the human diet. Its chemical structure is the basis of all flavonoids present in black and green tea, soybean, red fruits and so on. Although offering such nutritional value, it still requires a molecular approach to understand its interactions with a specific target. The combination of experimental and computational techniques makes it possible to describe the interaction between 2PHE and human serum albumin (HSA). Fluorescence spectroscopy results show that the quenching mechanism is static, and thermodynamic analysis points to an entropically driven complex. The binding density function method provides information about a positive cooperative interaction, while drug displacement experiments indicate Sites 1 and 2 of HSA as the most probable binding sites. From the molecular dynamic study, it appears that the molecular docking is in agreement with experimental data and thus more realistic.

Experimental evidence and molecular modeling of the interaction between hRSV-NS1 and quercetin.

Human Respiratory Syncytial Virus is one of the major causes of acute respiratory infections in children, causing bronchiolitis and pneumonia. Non-Structural Protein 1 (NS1) is involved in immune system evasion, a process that contributes to the success of hRSV replication. This protein can act by inhibiting or neutralizing several steps of interferon pathway, as well as by silencing the hRSV ribonucleoproteic complex. There is evidence that quercetin can reduce the infection and/or replication of several viruses, including RSV. The aims of this study include the expression and purification of the NS1 protein besides experimental and computational assays of the NS1-quercetin interaction. CD analysis showed that NS1 secondary structure composition is 30% alpha-helix, 21% beta-sheet, 23% turn and 26% random coils. The melting temperature obtained through DSC analysis was around 56°C. FRET analysis showed a distance of approximately 19Å between the NS1 and quercetin. Fluorescence titration results showed that the dissociation constant of the NS1-quercetin interaction was around 10(-6)M. In thermodynamic analysis, the enthalpy and entropy balanced forces indicated that the NS1-quercetin interaction presented both hydrophobic and electrostatic contributions. The computational results from the molecular modeling for NS1 structure and molecular docking regarding its interaction with quercetin corroborate the experimental data.

The interaction between heparin and Lys49 phospholipase A2 reveals the natural binding of heparin on the enzyme.

We have studied at a molecular level the interaction of heparins on bothropstoxin-I (BthTx-I), a phospholipase A2 toxin. The protein was monitored using gel filtration chromatography, dynamic light scattering (DLS), circular dichroism (CD), attenuated total reflectance Fourier transform infrared (ATR-FTIR) and intrinsic tryptophan fluorescence emission (ITFE) spectroscopy. The elution profile of the protein presents a displacement of the protein peak to larger complexes when interacting with higher concentration of heparin. The DLS results shows two Rh at a molar ratio of 1, one to the distribution of the protein and the second for the action of heparin on BthTx-I structures, and a large distribution with the increase of protein. The interaction is accompanied by significant changes in the CD spectra, showing two common features: a decrease in signal at 208 nm (3 and 6 kDa heparins) and an isodichroic point near 226 nm (3 kDa heparin). FTIR spectra indicate that only a few amino acid residues are involved in this interaction. Alterations in the ITFE by binding heparins suggest that the initial binding occurs on the ventral face of BthTx-I. Together, these results add an experimental and structural basis on the action mechanism of the heparins over the phospholipases A2 and provide a molecular model to elucidate the interaction of the enzyme-heparin complex at a molecular level.

Binding of antioxidant flavone isovitexin to human serum albumin investigated by experimental and computational assays.

The flavonoids are a large class of polyphenolic compounds which occur naturally in plants where they are widely distributed. Isovitexin (ISO) is a glycosylated flavonoid that exhibits a potential antioxidant activity. Some recent studies have shown the pharmacokinetic activity of isovitexin in rat blood plasma, however, without detailing the molecular target that is linked and what physicochemical forces govern the interaction. In mammalians, the most abundant protein in blood plasma is the albumin and is not unlike with human, which human serum albumin (HSA) is the major extracellular protein and functions as a carrier of various drugs. The interaction between HSA and ISO was investigated using fluorescence, UV-vis absorbance, circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR) together with, computational methods like ab initio and molecular modeling calculation. Fluorescence quenching indicated that ISO location is within the hydrophobic pocket in subdomain IIA (site 1) of HSA, close to the Trp214 residue. The Stern-Volmer quenching constants determined at 288, 298 and 308K and its dependence on temperature indicated that the quenching mechanism was static. From the analysis of binding equilibrium were determined; the binding site number and binding constants, with the correspondent thermodynamic parameters, ΔH, ΔG and ΔS for HSA-ISO complex. Also, a second binding analysis, binding density function (BDF) method, which is independent of any binding model pre-established obtained similar results. The fluorescence resonance energy transfer estimated the distance between the donor (HSA-Trp214) and acceptor (ISO), while FT-IR and CD spectroscopy measured possible changes of secondary structure at the formation of the HSA-ISO complex. The optimized geometry of isovitexin calculation performed with its ground state by using DFT/B3LYP/6-311+G(d,p) method. The HSA-ISO complex interactions determined by molecular modeling tool corroborated with the thermodynamic analysis from the experimental data.

Photoacoustic spectroscopy of aromatic amino acids in proteins.

This paper concerns the use of photoacoustic spectroscopy (PAS) to study the presence of aromatic amino acid in proteins. We examined the aromatic amino acids in six proteins with well-known structures using absorption spectra of near ultraviolet PAS over the wavelength range 240-320 nm. The fundamental understanding of the physical and chemical properties that govern the absorption of light and a subsequent release of heat to generate a transient pressure wave was used to test the concept of monitoring aromatic amino acids with this method. Second derivative spectroscopy in the ultraviolet region of proteins was also used to study the regions surrounding the aromatics and the percentage area in each band was related in order to determine the contribution in function of the respective molar extinction coefficients for each residue. Further investigation was conducted into the interaction between sodium dodecyl sulphate (SDS) and bothropstoxin-I (BthTx-I), with the purpose of identifying the aromatics that participate in the interaction. The clear changes in the second derivative and curve-fitting procedures suggest that initial SDS binding to the tryptophan located in the dimer interface and above 10 SDS an increased intensity between 260 and 320 nm, demonstrating that the more widespread tyrosine and phenylalanine residues contribute to the SDS/BthTx-I interactions. These results demonstrate the potential of near UV-PAS for the investigation of membrane proteins/detergent complexes in which light scattering is significant.

An ultraviolet photoacoustic spectroscopy study of the interaction between Lys49-phospholipase A2 and amphiphilic molecules.

We have used near ultraviolet photoacoustic spectroscopy (PAS) over the wavelength range 240-320 nm to investigate the complex formed between the homodimeric bothropstoxin-I, a lysine-49-phospholipase A2 from the venom of Bothrops jararacussu (BthTx-I), with the anionic amphiphile sodium dodecyl sulfate (SDS). At molar ratios>10, the complex developed a significant light scatter, accompanied by a decrease in the intrinsic tryptophan fluorescence intensity emission (ITFE) of the protein, and an increase in the near UV-PAS signal. Difference PAS spectroscopy at SDS/BthTx-I ratios<8 were limited to the region 280-290 nm, suggesting initial SDS binding to the tryptophan 77 located at the dimer interface. At SDS/BthTx-I ratios>10, the intensity between 260 and 320 nm increases demonstrating that the more widespread tyrosine and phenylalanine residues contribute to the SDS/BthTx-I interaction. PAS signal phase changes at wavelengths specific for each aromatic residue suggest that the Trp77 becomes more buried on SDS binding, and that protein structural changes and dehydration may alter the microenvironments of Tyr and Phe residues. These results demonstrate the potential of near UV-PAS for the investigation of membrane proteins/detergent complexes in which light scatter is significant.

A new biophysics approach using photoacoustic spectroscopy to study the DNA-ethidium bromide interaction.

We have examined the binding processes of ethidium bromide interacting with calf thymus DNA using photoacoustic spectroscopy. These binding processes are generally investigated by a combination of absorption or fluorescence spectroscopies with hydrodynamic techniques. The employment of photoacoustic spectroscopy for the DNA-ethidium bromide system identified two binding manners for the dye. The presence of two isosbestic points (522 and 498 nm) during DNA titration was evidence of these binding modes. Analysis of the photoacoustic amplitude signal data was performed using the McGhee-von Hippel excluded site model. The binding constant obtained was 3.4 x 10(8) M(bp)(-1), and the number of base pairs excluded to another dye molecule by each bound dye molecule (n) was 2. A DNA drug dissociation process was applied using sodium dodecyl sulfate to elucidate the existence of a second and weaker binding mode. The dissociation constant determined was 0.43 mM, whose inverse value was less than the previously obtained binding constant, demonstrating the existence of the weaker binding mode. The calculated binding constant was adjusted by considering the dissociation constant and its new value was 1.2 x 10(9) M(bp)(-1) and the number of excluded sites was 2.6. Using the photoacoustic technique it is also possible to obtain results regarding the dependence of the quantum yield of the dye on its binding mode. While intercalated between two adjacent base pairs the quantum yield found was 0.87 and when associated with an external site it was 0.04. These results reinforce the presence of these two binding processes and show that photoacoustic spectroscopy is more extensive than commonly applied spectroscopies.