Small molecule–biopolymer interactions : Ultraviolet–visible and fluorescence spectroscopy and chemometrics

Interactions between small molecules with biopolymers e.g. the bovine serum albumin (BSA protein), are important, and significant information is recorded in the UV–vis and fluorescence spectra of their reaction mixtures. The extraction of this information is difficult conventionally and principally because there is significant overlapping of the spectra of the three analytes in the mixture. The interaction of berberine chloride (BC) and the BSA protein provides an interesting example of such complex systems. UV–vis and fluorescence spectra of BC and BSA mixtures were investigated in pH 7.4 Tris–HCl buffer at 37 °C. Two sample series were measured by each technique: (1) [BSA] was kept constant and the [BC] was varied and (2) [BC] was kept constant and the [BSA] was varied. This produced four spectral data matrices, which were combined into one expanded spectral matrix. This was processed by the multivariate curve resolution–alternating least squares method (MCR–ALS). The results produced: (1) the extracted pure BC, BSA and the BC–BSA complex spectra from the measured heavily overlapping composite responses, (2) the concentration profiles of BC, BSA and the BC–BSA complex, which are difficult to obtain by conventional means, and (3) estimates of the number of binding sites of BC.

Integrin-functionalized artificial membranes as test platforms for monitoring small integrin ligand binding by surface plasmon-enhanced fluorescence spectroscopy

The design and synthesis of molecularly or supramolecularly defined interfacial architectures have seen in recent years a remarkable growth of interest and scientific research activities for various reasons. On the one hand, it is generally believed that the construction of an interactive interface between the living world of cells, tissue, or whole organisms and the (inorganic or organic) materials world of technical devices such as implants or medical parts requires proper construction and structural (and functional) control of this organism–machine interface. It is still the very beginning of generating a better understanding of what is needed to make an organism tolerate implants, to guarantee bidirectional communication between microelectronic devices and living tissue, or to simply construct interactive biocompatibility of surfaces in general. This exhaustive book lucidly describes the design, synthesis, assembly and characterization, and bio-(medical) applications of interfacial layers on solid substrates with molecularly or supramolecularly controlled architectures. Experts in the field share their contributions that have been developed in recent years.

Binding of small mono- and oligomeric integrin ligands to membrane-embedded integrins monitored by surface plasmon-enhanced fluorescence spectroscopy

We recently developed a binding assay format by incorporating native transmembrane receptors into artificial phospholipid bilayers on biosensor devices for surface plasmon resonance spectroscopy. By extending the method to surface plasmon-enhanced fluorescence spectroscopy (SPFS), sensitive recording of the association of even very small ligands is enabled. Herewith, we monitored binding of synthetic mono- and oligomeric RGD-based peptides and peptidomimetics to integrins alphavbeta3 and alphavbeta5, after having confirmed correct orientation and functionality of membrane-embedded integrins. We evaluated integrin binding of RGD multimers linked together via aminohexanoic acid (Ahx) spacers and showed that the dimer revealed higher binding activity than the tetramer, followed by the RGD monomers. The peptidomimetic was also found to be highly active with a slightly higher selectivity toward alphavbeta3. The different compounds were also evaluated in in vitro cell adhesion tests for their capacity to interfere with alphavbeta3-mediated cell attachment to vitronectin. We hereby demonstrated that the different RGD monomers were similarly effective; the RGD dimer and tetramer showed comparable IC50 values, which were, however, significantly higher than those of the monomers. Best cell detachment from vitronectin was achieved by the peptidomimetic. The novel SPFS-binding assay platform proves to be a suitable, reliable, and sensitive method to monitor the binding capacity of small ligands to native transmembrane receptors, here demonstrated for integrins.

Effect of handling and fixation processes on fluorescence spectroscopy of mouse skeletal muscles under two-photon excitation

We investigated the effects of handling and fixation processes on the two-photon fluorescence spectroscopy of endogenous fluorophors in mouse skeletal muscle. The skeletal muscle was handled in one of two ways: either sectioned without storage or sectioned following storage in a freezer. The two-photon fluorescence spectra measured for different storage or fixation periods show a differential among those samples that were stored in water or were fixed either in formalin or methanol. The spectroscopic results indicate that formalin was the least disruptive fixative, having only a weak effect on the two-photon fluorescence spectroscopy of muscle tissue, whereas methanol had a significant influence on one of the autofluorescence peaks. The two handling processes yielded similar spectral information, indicating no different effects between them.

Two-photon fluorescence spectroscopy for identification of healthy and malignant biological tissues

Two-photon fluorescence spectroscopy has been performed on rat skeletal muscles to investigate the effect of fixation processes on the micro-environments of the endogenous fluorophors in rat skeletal muscles. The two-photon fluorescence spectra measured for different fixation periods show a differential among those samples that were fixed in water, formalin and methanol, respectively. The results imply that two-photon fluorescence spectroscopy can be a potential technique for identification of healthy and malignant biological tissues.

Synchronous fluorescence and UV–vis spectroscopic studies of interactions between the tetracycline antibiotic, aluminium ions and DNA with the aid of the Methylene Blue dye probe

Synchronous fluorescence spectroscopy (SFS) was applied for the investigation of interactions of the antibiotic, tetracycline (TC), with DNA in the presence of aluminium ions (Al3+). The study was facilitated by the use of the Methylene Blue (MB) dye probe, and the interpretation of the spectral data with the aid of the chemometrics method, parallel factor analysis (PARAFAC). Three-way synchronous fluorescence analysis extracted the important optimum constant wavelength differences, Δλ, and showed that for the TC–Al3+–DNA, TC–Al3+ and MB dye systems, the associated Δλ values were different (Δλ = 80, 75 and 30 nm, respectively). Subsequent PARAFAC analysis demonstrated the extraction of the equilibrium concentration profiles for the TC–Al3+, TC–Al3+–DNA and MB probe systems. This information is unobtainable by conventional means of data interpretation. The results indicated that the MB dye interacted with the TC–Al3+–DNA surface complex, presumably via a reaction intermediate, TC–Al3+–DNA–MB, leading to the displacement of the TC–Al3+ by the incoming MB dye probe.

Fluorescence spectrometric study on the interactions of isoprocarb and sodium 2-isopropylphenate with bovine serum albumin

The binding interaction of the pesticide Isoprocarb and its degradation product, sodium 2-isopropylphenate, with bovine serum albumin (BSA) was studied by spectrofluorimetry under simulated physiological conditions. Both Isoprocarb and sodium 2-isopropylphenate quenched the intrinsic fluorescence of BSA. This quenching proceeded via a static mechanism. The thermodynamic parameters (ΔH°, ΔS° and ΔG°) obtained from the fluorescence data measured at two different temperatures showed that the binding of Isoprocarb to BSA involved hydrogen bonds and that of sodium 2-isopropylphenate to BSA involved hydrophobic and electrostatic interactions. Synchronous fluorescence spectroscopy of the interaction of BSA with either Isoprocarb or sodium 2-isopropylphenate showed that the molecular structure of the BSA was changed significantly, which is consistent with the known toxicity of the pesticide, i.e., the protein is denatured. The sodium 2-isopropylphenate, was estimated to be about 4–5 times more toxic than its parent, Isoprocarb. Synchronous fluorescence spectroscopy and the resolution of the three-way excitation–emission fluorescence spectra by the PARAFAC method extracted the relative concentration profiles of BSA, Isoprocab and sodium 2-isopropylphenate as a function of the added sodium 2-isopropylphenate. These profiles showed that the degradation product, sodium 2-isopropylphenate, displaced the pesticide in a competitive reaction with the BSA protein.

meso-Porphyrinylphosphine Oxides: Mono- and Bidentate Ligands for Supramolecular Chemistry and the Crystal Structures of Monomeric [10,20-Diphenylporphyrinatonickel(II)-5,15-diyl]-bis-[P(O)Ph2] and Polymeric Self-Coordinated ([10,20-Diphenylporphyrinatozinc(II)-5,15-diyl]-bis-[P(O)Ph2])

A series of porphyrins substituted in one or two meso-positions by diphenylphosphine oxide groups has been prepared by the palladium catalysed reaction of diphenylphosphine or its oxide with the corresponding bromoporphyrins. Compounds {MDPP-[P(O)Ph2]n} (M = H2, Ni, Zn; H2DPP = 5,15-diphenylporphyrin; n = 1, 2) were isolated in yields of 60-95%. The reaction is believed to proceed via the conventional oxidative addition, phosphination and reductive elimination steps, as the stoichiometric reaction of η1-palladio(II) porphyrin [PdBr(H2DPP)(dppe)] (H2DPP = 5,15-diphenylporphyrin; dppe = 1,2-bis(diphenylphosphino)ethane) with diphenylphosphine oxide also results in the desired mono-porphyrinylphosphine oxide [H2DPP-P(O)Ph2]. Attempts to isolate the tertiary phosphines failed due to their extreme air-sensitivity. Variable temperature 1H NMR studies of [H2DPP-P(O)Ph2] revealed an intrinsic lack of symmetry, while fluorescence spectroscopy showed that the phosphine oxide group does not behave as a "heavy atom" quencher. The electron withdrawing effect of the phosphine oxide group was confirmed by voltammetry. The ligands were characterised by multinuclear NMR and UV-visible spectroscopy as well as mass spectrometry. Single crystal X-ray crystallography showed that the bis(phosphine oxide) nickel(II) complex {[NiDPP-[P(O)Ph2]2} is monomeric in the solid state, with a ruffled porphyrin core and the two P=O fragments on the same side of the average plane of the molecule. On the other hand, the corresponding zinc(II) complex formed infinite chains through coordination of one Ph2PO substituent to the neighbouring zinc porphyrin through an almost linear P=O---Zn unit, leaving the other Ph2PO group facing into a parallel channel filled with disordered water molecules. These new phosphine oxides are attractive ligands for supramolecular porphyrin chemistry.

Photochemistry and chemometrics : an overview

Photochemistry has made significant contributions to our understanding of many important natural processes as well as the scientific discoveries of the man-made world. The measurements from such studies are often complex and may require advanced data interpretation with the use of multivariate or chemometrics methods. In general, such methods have been applied successfully for data display, classification, multivariate curve resolution and prediction in analytical chemistry, environmental chemistry, engineering, medical research and industry. However, in photochemistry, by comparison, applications of such multivariate approaches were found to be less frequent although a variety of methods have been used, especially with spectroscopic photochemical applications. The methods include Principal Component Analysis (PCA; data display), Partial Least Squares (PLS; prediction), Artificial Neural Networks (ANN; prediction) and several models for multivariate curve resolution related to Parallel Factor Analysis (PARAFAC; decomposition of complex responses). Applications of such methods are discussed in this overview and typical examples include photodegradation of herbicides, prediction of antibiotics in human fluids (fluorescence spectroscopy), non-destructive in- and on-line monitoring (near infrared spectroscopy) and fast-time resolution of spectroscopic signals from photochemical reactions. It is also quite clear from the literature that the scope of spectroscopic photochemistry was enhanced by the application of chemometrics. To highlight and encourage further applications of chemometrics in photochemistry, several additional chemometrics approaches are discussed using data collected by the authors. The use of a PCA biplot is illustrated with an analysis of a matrix containing data on the performance of photocatalysts developed for water splitting and hydrogen production. In addition, the applications of the Multi-Criteria Decision Making (MCDM) ranking methods and Fuzzy Clustering are demonstrated with an analysis of water quality data matrix. Other examples of topics include the application of simultaneous kinetic spectroscopic methods for prediction of pesticides, and the use of response fingerprinting approach for classification of medicinal preparations. In general, the overview endeavours to emphasise the advantages of chemometrics' interpretation of multivariate photochemical data, and an Appendix of references and summaries of common and less usual chemometrics methods noted in this work, is provided. Crown Copyright © 2010.

Mineralogy and Magnetic Properties of Basaltic Substrate Soils: Kaho'olawe and Big Island, Hawaii

Magnetic behavior of soils can seriously hamper the performance of geophysical sensors. Currently, we have little understanding of the types of minerals responsible for the magnetic behavior, as well as their distribution in space and evolution through time. This study investigated the magnetic characteristics and mineralogy of Fe-rich soils developed on basaltic substrate in Hawaii. We measured the spatial distribution of magnetic susceptibility (χlf) and frequency dependence (χfd%) across three test areas in a well-developed eroded soil on Kaho'olawe and in two young soils on the Big Island of Hawaii. X-ray diffraction spectroscopy, x-ray fluorescence spectroscopy (XFCF), chemical dissolution, thermal analysis, and temperature-dependent magnetic studies were used to characterize soil development and mineralogy for samples from soil pits on Kaho'olawe, surface samples from all three test areas, and unweathered basalt from the Big Island of Hawaii. The measurements show a general increase in magnetic properties with increasing soil development. The XRF Fe data ranged from 13% for fresh basalt and young soils on the Big Island to 58% for material from the B horizon of Kaho'olawe soils. Dithionite-extractable and oxalate-extractable Fe percentages increase with soil development and correlate with χlf-and χfd%, respectively. Results from the temperature-dependent susceptibility measurements show that the high soil magnetic properties observed in geophysical surveys in Kaho'olawe are entirely due to neoformed minerals. The results of our studies have implications for the existing soil survey of Kaho'olawe and help identify methods to characterize magnetic minerals in tropical soils.

Competitive interactions of anti-carcinogens with serum albumin: A spectroscopic study of bendamustine and dexamethasone with the aid of chemometrics

Interactions between the anti-carcinogens, bendamustine (BDM) and dexamethasone (DXM), with bovine serum albumin (BSA) were investigated with the use of fluorescence and UV–vis spectroscopies under pseudo-physiological conditions (Tris–HCl buffer, pH 7.4). The static mechanism was responsible for the fluorescence quenching during the interactions; the binding formation constant of the BSA–BDM complex and the binding number were 5.14 × 105 L mol−1 and 1.0, respectively. Spectroscopic studies for the formation of BDM–BSA complex were interpreted with the use of multivariate curve resolution – alternating least squares (MCR–ALS), which supported the complex formation. The BSA samples treated with site markers (warfarin – site I and ibuprofen – site II) were reacted separately with BDM and DXM; while both anti-carcinogens bound to site I, the binding constants suggested that DXM formed a more stable complex. Relative concentration profiles and the fluorescence spectra associated with BDM, DXM and BSA, were recovered simultaneously from the full fluorescence excitation–emission data with the use of the parallel factor analysis (PARAFAC) method. The results confirmed that on addition of DXM to the BDM–BSA complex, the BDM was replaced and the DXM–BSA complex formed; free BDM was released. This finding may have consequences for the transport of these drugs during any anti-cancer treatment.

Analysis of complex molecular systems: The impact of multivariate analysis for resolving the interactions of small molecules with biopolymers - a review

This review is focused on the impact of chemometrics for resolving data sets collected from investigations of the interactions of small molecules with biopolymers. These samples have been analyzed with various instrumental techniques, such as fluorescence, ultraviolet–visible spectroscopy, and voltammetry. The impact of two powerful and demonstrably useful multivariate methods for resolution of complex data—multivariate curve resolution–alternating least squares (MCR–ALS) and parallel factor analysis (PARAFAC)—is highlighted through analysis of applications involving the interactions of small molecules with the biopolymers, serum albumin, and deoxyribonucleic acid. The outcomes illustrated that significant information extracted by the chemometric methods was unattainable by simple, univariate data analysis. In addition, although the techniques used to collect data were confined to ultraviolet–visible spectroscopy, fluorescence spectroscopy, circular dichroism, and voltammetry, data profiles produced by other techniques may also be processed. Topics considered including binding sites and modes, cooperative and competitive small molecule binding, kinetics, and thermodynamics of ligand binding, and the folding and unfolding of biopolymers. Applications of the MCR–ALS and PARAFAC methods reviewed were primarily published between 2008 and 2013.