How triacylglycerol thermal history impacts film removal by surfactant solution.

HYPOTHESIS: The physical and mechanical properties of triacylglycerols (TAGs), or 'fats', depend on their composition and thermal history which, in turn, impact crystal structure and morphology. We examine whether thermal history can be mechanistically related to film removal by a surfactant solution. EXPERIMENTS: Model TAG mixtures, comprising triolein:tripalmitin:tristearin 0.5:0.3:0.2, were subjected to a range of cooling profiles from the melt (0.5-80°C/min, Newtonian and annealed), and the resulting solid films characterised by microscopy, X-ray diffraction, infrared spectroscopy, and contact angle measurements. Film removal from a model glass substrate by an aqueous surfactant solution of sodium dodecylsulphate and dodecyldimethylamine oxide at room temperature fixed at 25°C was examined under quiescent flow conditions. FINDINGS: Quantitative relations are established between TAG cooling profile, crystal structure and morphology, surface energy γSFE, and removal (or 'cleaning'). In general, films cooled slowly from the melt yield heterogeneous morphologies with predominantly ß1' phase, higher polar γSFE, and faster removal timescales. By contrast, rapid cooling results in homogeneous films, rich in ß2' phase, low polar γSFE, and long removal times. Our results elucidate the non-trivial impact of TAG thermal history, connecting the multiscale semi-crystalline structure to surface energy, and eventually to film delamination by micellar solutions.

Concentration Dependent Asymmetric Synergy in SDS-DDAO Mixed Surfactant Micelles.

We investigate the structure and interactions of a model anionic/amphoteric mixed surfactant micellar system, namely, sodium dodecyl sulfate (SDS) and N,N-dimethyldodecylamine N-oxide (DDAO), employing SANS, FTIR, DLS, and pH measurements, in the range 0.1-100 mM total surfactant concentration and 0-100% DDAO. Increasing surfactant concentration is found to elongate the prolate ellipsoid micelles (RPolar ∼ 25-40 Å), accompanied by up to a 6-fold increase in micellar charge. The surfactant synergy, in terms of micellar charge and size, diffusion coefficient, solution pH, and headgroup interactions, was found to vary with concentration. At lower concentrations (≤50 mM), the SDS-DDAO ratio of maximum synergy is found to be asymmetric (at 65-85% DDAO), which is rationalized using regular solution theory, suggesting an equilibrium between Na+ dissociation, DDAO protonation, and counterion concentration. At higher concentrations, maximum synergy shifts toward the equimolar ratio. Overall, our study expands and unifies previous reports, providing a comprehensive understanding for this model, synergetic mixed micellar system.

Phase behaviour of model triglyceride ternary blends: triolein, tripalmitin and tristearin.

We investigate the phase behavior of model ternary triacylglycerol blends, comprising triolein (C57H104O6, OOO), tripalmitin (C51H98O6, PPP) and tristearin (C57H110O6, SSS), building upon extensive characterisation of single and binary mixtures, in order to rigorously map the thermal transitions of model natural 'fats'. A combination of calorimetry, X-ray diffraction, and FTIR spectroscopy is employed to determine crystallisation and melting temperatures and identify the corresponding phases in the complex ternary system. We recover the eutectic behaviour of SSS-PPP blends and the invariability of OOO neat transitions, and resolve the complex ß' + ß ternary surface, reflecting the roles of unsaturation and polymorphism of its constituents. Our results provide a representation of the OOO:PPP:SSS:temperature phase behaviour into a triangular prism, consistent with binary pair-wise data, which can inform a range of food science, cosmetic, pharmaceutical and cleaning applications that depend strongly on the physical-chemistry of such multicomponent 'triglycerides'.

Solution Structures of Anionic-Amphoteric Surfactant Mixtures near the Two-Phase Region at Fixed pH.

We examine the solution structures in a mixed surfactant system of sodium dodecyl sulfate (SDS) and N,N-dimethyldodecylamine N-oxide (DDAO) in water, on both sides of the two-phase boundary, employing dynamic light scattering, small-angle neutron scattering, and Fourier transform infrared spectroscopy. The precipitate phase boundary was accessed by lowering pH to 8, from its floating pH 9.5 value, and was experimentally approached from the monomeric and micellar regions in three ways: at fixed DDAO or SDS concentrations and at a fixed (70:30) SDS:DDAO molar ratio. We characterize the size, shape, and interactions of micelles, which elongate approaching the boundary, leading to the formation of disk-like aggregates within the biphasic region, coexisting with micelles and monomers. Our data, from both monomeric and micellar solutions, indicate that the two phase structures formed are largely pathway-independent, with dimensions influenced by both pH and mixed surfactant composition. Precipitation occurs at intermediate stoichiometries with a similar SDS:DDAO ratio, whereas asymmetric stoichiometries form a re-entrant transition, returning to the mixed micelle phase. Overall, our findings demonstrate the effect of stoichiometry and solution pH on the synergistic interaction of mixed surfactants and their impact on phase equilibrium and associated micellar and two-phase structures.

Light Responsiveness and Assembly of Arylazopyrazole-Based Surfactants in Neat and Mixed CTAB Micelles.

The self-assembly of an arylazopyrazole-based photosurfactant (PS), based on cetyltrimethylammonium bromide (CTAB), and its mixed micelle formation with CTAB in aqueous solution was investigated by small angle neutron and X-ray scattering (SANS/SAXS) and UV-vis absorption spectroscopy. Upon UV light exposure, PS photoisomerizes from E-PS (trans) to Z-PS (cis), which transforms oblate ellipsoidal micelles into smaller, spherical micelles with larger shell thickness. Doping PS with CTAB resulted in mixed micelle formation at all stoichiometries and conditions investigated; employing selectively deuterated PS, a monotonic variation in scattering length density and dimensions of the micellar core and shell is observed for all contrasts. The concentration- and irradiance-dependence of the E to Z configurational transition was established in both neat and mixed micelles. A liposome dye release assay establishes the enhanced efficacy of photosurfactants at membrane disruption, with E-PS exhibiting a 4-fold and Z-PS a 10-fold increase in fluorescence signal with respect to pure CTAB. Our findings pave the way for external triggering and modulation of the wide range of CTAB-based biomedical and material applications.

Ionotropic Gelation Fronts in Sodium Carboxymethyl Cellulose for Hydrogel Particle Formation.

Hydrogel microparticles (HMPs) find numerous practical applications, ranging from drug delivery to tissue engineering. Designing HMPs from the molecular to macroscopic scales is required to exploit their full potential as functional materials. Here, we explore the gelation of sodium carboxymethyl cellulose (NaCMC), a model anionic polyelectrolyte, with Fe3+ cations in water. Gelation front kinetics are first established using 1D microfluidic experiments, and effective diffusive coefficients are found to increase with Fe3+ concentration and decrease with NaCMC concentrations. We use Fourier Transform Infrared Spectroscopy (FTIR) to elucidate the Fe3+-NaCMC gelation mechanism and small angle neutron scattering (SANS) to spatio-temporally resolve the solution-to-network structure during front propagation. We find that the polyelectrolyte chain cross-section remains largely unperturbed by gelation and identify three hierarchical structural features at larger length scales. Equipped with the understanding of gelation mechanism and kinetics, using microfluidics, we illustrate the fabrication of range of HMP particles with prescribed morphologies.

Surface-Induced Crystallization of Sodium Dodecyl Sulfate (SDS) Micellar Solutions in Confinement.

We investigate the role of confinement on the onset of crystallization in subcooled micellar solutions of sodium dodecyl sulfate (SDS), examining the impact of sample volume, substrate surface energy, and surface roughness. Using small angle neutron scattering (SANS) and dynamic light scattering (DLS), we measure the crystallization temperature upon cooling and the metastable zone width (MSZW) for bulk 10-30 wt% SDS solutions. We then introduce a microdroplet approach to quantify the impact of surface free energy (18-65 mN/m) and substrate roughness (Rα ≃ 0-60 µm) on the kinetics of surface-induced crystallization through measurements of induction time (ti) under isothermal conditions. While ti is found to decrease exponentially with decreasing temperature (increasing subcooling) for all tested surfaces, increasing the surface energy could cause a significant further reduction of up to ∼40 fold. For substrates with the lowest surface energy and longest ti, microscale surface roughness is found to enhance crystal nucleation, in particular for Rα ≥ 10 µm. Finally, we demonstrate that tuning the surface energy and microscopic roughness can be effective routes to promote or delay nucleation in bulk-like volumes, thus greatly impacting the stability of surfactant solutions at lower temperatures.

Pure and mixed aqueous micellar solutions of Sodium Dodecyl sulfate (SDS) and Dimethyldodecyl Amine Oxide (DDAO): Role of temperature and composition.

Aqueous mixtures of anionic and nonionic/cationic surfactants can form non-trivial self-assemblies in solution and exhibit macroscopic responses. Here, we investigate the micellar phase of pure and mixed aqueous solutions of Sodium Dodecyl Sulfate (SDS) and Dimethyldodecyl Amine Oxide (DDAO) using a combination of Small Angle Neutron Scattering (SANS), Fourier-Transform Infrared Spectroscopy (FTIR) and rheological measurements. We examine the effect of temperature (0-60 °C), on the 20 wt% SDS micellar solutions with varying DDAO (⩽5 wt%), and seek to correlate micellar structure with zero-shear solution viscosity. SANS establishes the formation of prolate ellipsoidal micelles in aqueous solutions of pure SDS, DDAO and SDS/DDAO mixtures, whose axial ratio is found to increase upon cooling. Elongation of the ellipsoidal micelles of pure SDS is also induced by the introduction of the non-anionic DDAO, which effectively reduces the repulsive interactions between the anionic SDS head-groups. In FTIR measurements, the formation of elongated mixed ellipsoidal micelles is confirmed by the increase of ordering in the hydrocarbon chain tails and interaction between surfactant head-groups. We find that the zero-shear viscosity of the mixed surfactant solutions increases exponentially with decreasing temperature and increasing DDAO content. Significantly, a master curve for solution viscosity can be obtained in terms of micellar aspect ratio, subsuming the effects of both temperature and DDAO composition in the experimental range investigated. The intrinsic viscosity of mixed micellar solutions is significantly larger than the analytical and numerical predictions for Brownian suspensions of ellipsoidal colloids, highlighting the need to consider interactions of soft micelles under shear, especially at high concentrations.

Salivary Raman Spectroscopy: Standardization of Sampling Protocols and Stratification of Healthy and Oral Cancer Subjects.

Minimally invasive cancer detection using bio-fluids has been actively pursued due to practical limitations, though there are better suited noninvasive and online in vivo methods. Saliva is one such clinically informative bio-fluid that offers the advantages of easy and multiple sample collection. Despite its potential in cancer diagnostics, saliva analysis is challenging due to its heterogeneous composition. Recently, there has been an upsurge in saliva exploration using optical techniques. Forms of saliva such as precipitate and supernatant have been monitored, but this sampling method needs to be standardized due to the obvious loss of analytes in processing. In that context, present work details the comparison of four different saliva sampling methodologies, i.e., air-dried, lyophilized, pellet, and supernatant using Raman spectroscopy collected from 10 healthy samples. Composition-driven spectral features of all forms were compared and classified using principal component analysis and linear discriminant analysis. Analysis was carried out on all four groups in the first step. In the second step, groups of pellet and supernatant , and air-dried and lyophilized were analyzed. Findings suggest that pellet and supernatant exhibit discrete spectroscopic features and demonstrate high classification efficiency, which is indicative of their distinctive biochemical composition. On the other hand, air-dried and lyophilized forms showed overlapping spectral features and low classification, suggesting these forms retain majority spectroscopic features of whole saliva and are less prone to sampling losses. Thus, this study indicates air-dried and lyophilized forms may be more appropriate for saliva sampling using Raman spectroscopy providing the comprehensive information required for cancer diagnosis. Furthermore, the method was also tested for the classification of oral cancer and healthy subjects (n = 27) which yielded 90% stratification. The findings of the study indicate the utility of minimally invasive salivary Raman-based diagnostics in oral cancers.

Allicin Overcomes Hypoxia Mediated Cisplatin Resistance in Lung Cancer Cells through ROS Mediated Cell Death Pathway and by Suppressing Hypoxia Inducible Factors.

BACKGROUND/AIMS: The hypoxic microenvironment in NSCLC has been widely accepted as a contributor to both therapeutic resistance and tumor progression. In this study, we have explored Allicin, a key organosulfur compound present in garlic for its previously unreported effectiveness in the heterogeneous hypoxic tumor microenvironment of NSCLC. METHODS: The effect of Allicin on the viability of NSCLC cells was determined by MTT assay. To determine the migration rate of treated cells compared to the control, scratch and transwell migration assays were performed. Flowcytometry was done to explore cell cycle distribution, apoptosis and ROS production in cells. Fluorescence microscopy was used to examine autophagy and DNA damage in cells. Dot blot was done to check genome wide methylation. RNA expression was detected by RT-PCR and protein expression by western blotting. RESULTS: Allicin significantly decreases cell viability, proliferation and migration of NSCLC cells in both normoxia and hypoxia. It elicits both apoptosis and autophagy pathway in A549 cells by ROS accumulation and facilitating S/G2-M phase arrest in both normoxia as well as hypoxia. We suggest that ROS/MAPK and ROS/JNK signaling pathway together govern the cytotoxic effect of allicin in NSCLC cells. Notably, allicin suppresses the expression of HIF-1α and HIF-2α in hypoxic cells, pointing towards a mechanism of its effectiveness in hypoxia. A long term passive demethylation was observed, with decreased mC and no change in TET expression, thereby ruling out active demethylation by allicin. Furthermore, allicin synergistically enhances growth inhibitory activity of low dose cisplatin to effectively overcome hypoxia induced cisplatin resistance in A549 cells. CONCLUSION: Altogether, our results elucidate a potential use of allicin in sensitizing hypoxic and chemoresistant NSCLC to cisplatin-based chemotherapy and provide new, affordable therapeutic strategy with reduced side effects.

Raman exfoliative cytology for oral precancer diagnosis.

Oral premalignant lesions (OPLs) such as leukoplakia, erythroplakia, and oral submucous fibrosis, often precede oral cancer. Screening and management of these premalignant conditions can improve prognosis. Raman spectroscopy has previously demonstrated potential in the diagnosis of oral premalignant conditions (in vivo), detected viral infection, and identified cancer in both oral and cervical exfoliated cells (ex vivo). The potential of Raman exfoliative cytology (REC) in identifying premalignant conditions was investigated. Oral exfoliated samples were collected from healthy volunteers (n=20), healthy volunteers with tobacco habits (n=20), and oral premalignant conditions (n=27, OPL) using Cytobrush. Spectra were acquired using Raman microprobe. Spectral acquisition parameters were: λex: 785 nm, laser power: 40 mW, acquisition time: 15 s, and average: 3. Postspectral acquisition, cell pellet was subjected to Pap staining. Multivariate analysis was carried out using principal component analysis and principal component-linear discriminant analysis using both spectra- and patient-wise approaches in three- and two-group models. OPLs could be identified with ∼77% (spectra-wise) and ∼70% (patient-wise) sensitivity in the three-group model while with 86% (spectra-wise) and 83% (patient-wise) in the two-group model. Use of histopathologically confirmed premalignant cases and better sampling devices may help in development of improved standard models and also enhance the sensitivity of the method. Future longitudinal studies can help validate potential of REC in screening and monitoring high-risk populations and prognosis prediction of premalignant lesions.

Structural-conformational aspects of tRNA complexation with chloroethyl nitrosourea derivatives: A molecular modeling and spectroscopic investigation.

Chloroethyl nitrosourea derivatives (CENUs) represent an important family of anticancer chemotherapeutic agents, which are used in the treatment of different types of cancer such as brain tumors, resistant or relapsed Hodgkin's disease, small cell lung cancer and malignant melanoma. This work focuses towards understanding the interaction of chloroethyl nitrosourea derivatives; lomustine, nimustine and semustine with tRNA using spectroscopic approach in order to elucidate their auxiliary anticancer action mechanism inside the cell. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), Fourier transform infrared difference spectroscopy, circular dichroism spectroscopy and UV-visible spectroscopy were employed to investigate the binding parameters of tRNA-CENUs complexation. Results of present study demonstrate that all CENUs, studied here, interact with tRNA through guanine nitrogenous base residues and possibly further crosslink cytosine residues in paired region of tRNA. Moreover, spectral data collected for nimustine-tRNA and semustine-tRNA complex formation indicates towards the groove-directed-alkylation as their anti-malignant action, which involves the participation of uracil moiety located in major groove of tRNA. Besides this, tRNA-CENUs adduct formation did not alter the native conformation of biopolymer and tRNA remains in A-form after its interaction with all three nitrosourea derivatives studied. The binding constants (Ka) estimated for tRNA complexation with lomustine, nimustine and semustine are 2.55×102M-1, 4.923×102M-1 and 4.223×102M-1 respectively, which specify weak type of CENU's binding with tRNA. Moreover, molecular modeling simulations were also performed to predict preferential binding orientation of CENUs with tRNA that corroborates well with spectral outcomes. The findings, presented here, recognize tRNA binding properties of CENUs that can further help in rational designing of more specific and efficient RNA targeted chemotherapeutic agents.

tRNA binding with anti-cancer alkaloids-nature of interaction and comparison with DNA-alkaloids adducts.

Vincristine and vinblastine are potent anti-proliferative compound whose mechanism of action inside a cell is not well elucidated and the basis of their differential cellular effect is also unknown. This work focuses towards understanding the interaction of vincristine and vinblastine with tRNA using spectroscopic approach. Fourier transform infrared (FTIR) spectroscopy, Fourier transform infrared difference spectroscopy and UV-visible spectroscopy were used to study the binding parameters of tRNA-alkaloids interaction. Both the vinca alkaloids interact with tRNA through external binding with some degree of intercalation into the nitrogenous bases. The alkaloids adduct formation did not alter the A-conformation of the biopolymer and vincristine-tRNA complexes were found to be more stable than that of vinblastine-tRNA complexes. The binding constants (K) estimated for VCR-tRNA and VBS-tRNA complexation are 3×10(2)M(-1) and 2.5×10(2)M(-1) respectively, which suggests low affinity of alkaloids to tRNA. The study recognizes tRNA binding properties of vital vinca alkaloids and contributes to a better understanding of their mechanism of action and could also help in identifying the reason behind their diverse action in a cell.

Rapid determination of main constituents of packed juices by reverse phase-high performance liquid chromatography: an insight in to commercial fruit drinks.

The present work reports the compositional analysis of thirteen different packed fruit juices using high performance liquid chromatography (HPLC). Vitamin C, organic acids (citric and malic) and sugars (fructose, glucose and sucrose) were separated, analyzed and quantified using different reverse phase methods. A new rapid reverse phase HPLC method was developed for routine analysis of vitamin C in fruit juices. The precision results of the methods showed that the relative standard deviations of the repeatability and reproducibility were <0.05 and <0.1 respectively. Correlation coefficient of the calibration models developed was found to be higher than 0.99 in each case. It has been found that the content of Vitamin C was less variable amongst different varieties involved in the study. It is also observed that in comparison to fresh juices, the packed juices contain lesser amounts of vitamin C. Citric acid was found as the major organic acids present in packed juices while maximum portion of sugars was of sucrose. Comparison of the amount of vitamin C, organic acids and sugars in same fruit juice of different commercial brands is also reported.

Nucleic acid binding properties of allicin: spectroscopic analysis and estimation of anti-tumor potential.

BACKGROUND: Allicin has received much attention due to its anti-proliferative activity and not-well elucidated underlying mechanism of action. This work focuses towards determining the cellular toxicity of allicin and understanding its interaction with nucleic acid at molecular level. METHODS: MTT assay was used to assess the cell viability of A549 lung cancer cells against allicin. Fourier transform infrared (FTIR) and UV-visible spectroscopy were used to study the binding parameters of nucleic acid-allicin interaction. RESULTS: Allicin inhibits the proliferation of cancer cells in a concentration dependent manner. FTIR spectroscopy exhibited that allicin binds preferentially to minor groove of DNA via thymine base. Analysis of tRNA allicin complex has also revealed that allicin binds primarily through nitrogenous bases. Some amount of external binding with phosphate backbone was also observed for both DNA and RNA. UV visible spectra of both DNA allicin and RNA allicin complexes showed hypochromic shift with an estimated binding constant of 1.2×10(4)M(-1) for DNA and 1.06×10(3)M(-1)for RNA binding. No major transition from the B-form of DNA and A-form of RNA is observed after their interaction with allicin. CONCLUSIONS: The results demonstrated that allicin treatment inhibited the proliferation of A549 cells in a dose-dependent manner. Biophysical outcomes are suggestive of base binding and helix contraction of nucleic acid structure upon binding with allicin. GENERAL SIGNIFICANCE: The results describe cytotoxic potential of allicin and its binding properties with cellular nucleic acid, which could be helpful in deciphering the complete mechanism of cell death exerted by allicin.

Spectroscopic and molecular docking studies on chlorambucil interaction with DNA.

Chlorambucil (CMB) is an anticancer drug used for the treatment of variety of cancers. Structural and conformational changes associated with DNA after binding with CMB were explored using spectroscopic techniques to get insight into the mechanism of action of CMB at molecular level. Different molar ratios of CMB-DNA complex were prepared with constant DNA concentration under physiological conditions. FTIR spectroscopy, UV-visible spectroscopy, CD spectroscopy and molecular docking studies were employed to determine the binding site and binding constant of CMB with DNA. The results show CMB binds DNA through nitrogenous bases (thymine, guanine and cytosine). The binding constant was calculated to be 1.3 × 10³ M⁻¹, which suggests weak binding of CMB with DNA double helix. FTIR and CD results show that CMB do not disturb native B-conformation of DNA and it continues to remain in its B conformation even at higher concentrations of CMB. The molecular docking results are in corroboration with our experimental results and provides structural insight into the interaction site.

Binding of an indole alkaloid, vinblastine to double stranded DNA: a spectroscopic insight in to nature and strength of interaction.

Vinblastine is a chemotherapeutic drug, used for the treatment of various cancers. It functions by interfering with DNA in fast growing cells and preventing them from reproducing. The present work is focused on the interaction of vinblastine with double stranded DNA in aqueous solution. Fourier transform infrared and UV-Visible absorption spectroscopy were used to analyze the interaction of vinblastine with calf-thymus DNA. FTIR analysis showed binding of vinblastine through A-T and G-C base pairs of DNA along with its phosphate backbone. UV-Vis spectroscopy results suggested the intercalation of drug in between the base pairs of DNA double helix. The binding constant estimated for vinblastine-DNA association was found to be K=1.7×10(3)M(-1). Molecular docking was performed and the results showed adenine base binding of vinblastine with DNA. Furthermore spectroscopic results revealed that formation of vinblastine-DNA complex resulted in no major change in the B-conformation of DNA.

Analysis of ovarian tumor pathology by Fourier Transform Infrared Spectroscopy.

BACKGROUND: Ovarian cancer is the second most common cancer among women and the leading cause of death among gynecologic malignancies. In recent years, infrared (IR) spectroscopy has gained attention as a simple and inexpensive method for the biomedical study of several diseases. In the present study infrared spectra of normal and malignant ovarian tissues were recorded in the 650 cm-1 to 4000 cm-1 region. METHODS: Post surgical tissue samples were taken from the normal and tumor sections of the tissue. Fourier Transform Infrared (FTIR) data on twelve cases of ovarian cancer with different grades of malignancy from patients of different age groups were analyzed. RESULTS: Significant spectral differences between the normal and the ovarian cancerous tissues were observed. In particular changes in frequency and intensity in the spectral region of protein, nucleic acid and lipid vibrational modes were observed. It was evident that the sample-to-sample or patient-to-patient variations were small and the spectral differences between normal and diseased tissues were reproducible. CONCLUSION: The measured spectroscopic features, which are the spectroscopic fingerprints of the tissues, provided the important differentiating information about the malignant and normal tissues. The findings of this study demonstrate the possible use of infrared spectroscopy in differentiating normal and malignant ovarian tissues.

DNA interaction studies of an anticancer plant alkaloid, vincristine, using Fourier transform infrared spectroscopy.

The binding of vincristine with DNA has been investigated using Fourier transform infrared spectroscopy. Various changes in the double helical structure of DNA after addition of vincristine have been examined. It is evident from Fourier transform infrared results that vincristine-DNA interaction occurs through guanine and cytosine base pairs. External binding of vincristine with phosphate backbone of the DNA is also observed. Vincristine perturbs guanine band at 1714 cm(-1), cytosine band at 1488 cm(-1), and the phosphate vibrations at 1225 and 1086 cm(-1). The UV-visible spectra of vincristine-DNA complex show hypochromic and bathochromic shifts, indicating the intercalation of vincristine into the double helical structure of DNA. Both intercalative and external binding modes are observed for vincristine binding with DNA, with an estimated binding constant K = 1.0 × 10(3) M(-1).