Chlorophyll a Covalently Bonded to Organo-Modified Translucent Silica Xerogels: Optimizing Fluorescence and Maximum Loading.

Chlorophyll is a pyrrolic pigment with important optical properties, which is the reason it has been studied for many years. Recently, interest has been rising with respect to this molecule because of its outstanding physicochemical properties, particularly applicable to the design and development of luminescent materials, hybrid sensor systems, and photodynamic therapy devices for the treatment of cancer cells and bacteria. More recently, our research group has been finding evidence for the possibility of preserving these important properties of substrates containing chlorophyll covalently incorporated within solid pore matrices, such as SiO2, TiO2 or ZrO2 synthesized through the sol-gel process. In this work, we study the optical properties of silica xerogels organo-modified on their surface with allyl and phenyl groups and containing different concentrations of chlorophyll bonded to the pore walls, in order to optimize the fluorescence that these macrocyclic species displays in solution. The intention of this investigation was to determine the maximum chlorophyll a concentration at which this molecule can be trapped inside the pores of a given xerogel and to ascertain if this pigment remains trapped as a monomer, a dimer, or aggregate. Allyl and phenyl groups were deposited on the surface of xerogels in view of their important effects on the stability of the molecule, as well as over the fluorescence emission of chlorophyll; however, these organic groups allow the trapping of either chlorophyll a monomers or dimers. The determination of the above parameters allows finding the most adequate systems for subsequent in vitro or in vivo studies. The characterization of the obtained xerogels was performed through spectroscopic absorption, emission and excitation spectra. These hybrid systems can be employed as mimics of natural systems; the entrapment of chlorophyll inside pore matrices indicates that it is possible to exploit some of the most physicochemical properties of trapped chlorophyll for diverse technological applications. The data herein collected suggest the possibility of applying the developed methodology to other active, captive molecules in order to synthesize new hybrid materials with optimized properties, suitable to be applied in diverse technological fields.

Fluorescence and Textural Characterization of Ortho-Amine Tetraphenylporphyrin Covalently Bonded to Organo-Modified Silica Xerogels.

Most of the studies performed with porphyrins involve these species functionalized with peripheral substituents lying on the same macrocyclic molecular plane. The main objective of this work deals with the successful preservation and optimization of the fluorescence of a uncommonly used porphyrin species, i.e. tetrakis-(ortho-amino-phenyl)-porphyrin; a molecule with substituents localized not only at one but at both sides of its molecular plane. In cases like this, it must be stressed that fluorescence can only be partially preserved; nevertheless, intense fluorescence can still be reached by following a twofold functionalization strategy involving: (i) the bonding of substituted macrocycles to the pore walls of (ii) organo-modified silica monoliths synthesized by the sol-gel method. The analysis of both absorption and emission UV spectra evidenced a radiation energy transfer taking place between the porphyrin and the host silica matrix. Our results showed that the adequate displaying of the optical properties of macrocyclic species trapped in SiO2 xerogels depend on the polarity existing inside the pores, a property which can be tuned up through the adequate selection of organic groups used to modify the surface of the pore cavities. Additionally, the pore widths attained in the final xerogels can vary depending on the identity of the organic groups attached to the network. All these facts finally demonstrated that, even if using inefficient surface functionalization species, such as ortho-substituted tetraphenylporphyrins, it is still possible to modulate the pore shape, pore size, and physicochemical environment created around the trapped macrocycles. The most important aspect related to this research deals with the fact that the developed methodology offers a real possibility of controlling both the textural and morphological characteristics of a new kind of hybrid porous materials and to optimize the physicochemical properties of diverse active molecules trapped inside the pores of these materials.

Thermal denaturation of porcine pepsin: a study by circular dichroism.

Thermal denaturation of porcine pepsin in 10% ethanol was studied by circular dichroism (CD) spectroscopy. It was observed that the process is markedly irreversible. The denaturation unfolding process was strongly dependent on the heating rate, as is expected for an unfolding process kinetically controlled due to the presence of an irreversible reaction. Experimentally, we demonstrate the existence of an unfolded (U) state in equilibrium with the native (N) state. The U state is observed to exist at temperatures lower than 45 degrees C. The van't Hoff enthalpy, DeltaH(vH), was determined from direct estimation of the equilibrium constant at several temperatures (DeltaH(vH)=304.3 kJ/mol). To explain the observed behavior, we have considered a Lumry-Eyring model, which takes into account the presence of the U state in addition to N and denatured (D) states (i.e. N<-->U-->D).

Effect of irreversibility on the thermodynamic characterization of the thermal denaturation of Aspergillus saitoi acid proteinase.

The thermal denaturation of the acid proteinase from Aspergillus saitoi was studied by CD and differential scanning calorimetry (DSC). This process seemed to be completely irreversible, as protein samples that were heated to temperatures at which the transition had been completed and then cooled at 25 degrees C did not show any reversal of the change in the CD signal. Similar results were obtained with DSC. Nevertheless, we were able to detect the presence of reversibly unfolded species in experiments in which the enzyme solution was heated to a temperature within the transition region, followed by rapid cooling at 25 degrees C. Accordingly, the denaturation of behaviour of the acid proteinase seems to be consistent with the existence of one (or more) reversible unfolding transition followed by an irreversible step. The van't Hoff enthalpy, delta HvH, which corresponds to the reversible transition was calculated from extrapolation to infinite heating rate as 310 kJ.mol-1. This parameter was also determined from direct estimation of the equilibrium constant at several temperatures (delta HvH = 176 kJ.mol-1). Comparison of the average delta HvH with the calorimetric enthalpy (delta Hcal. = 770 kJ.mol-1) gave a value of 3.2 for the delta Hcal./delta HvH ratio, indicating that the molecular structure of the enzyme is probably formed by three or four cooperative regions, a number similar to that of the acid proteinase, pepsin. It should be noted that a completely different conclusion would be obtained from a straightforward analysis of the calorimetric curves, disregarding the effect of irreversibility on the denaturation process.

Circular dichroism studies of acid proteinases from Aspergillus niger and Aspergillus awamori.

Acid proteinases produced by strains of Aspergillus niger and Aspergillus awamori were isolated by means of ethanol precipitation, gel filtration and anion-exchange high resolution chromatography. In each case, the purified proteinase showed a single band in polyacrylamide gel electrophoresis. Their molecular weights were almost identical (approx. 45,000). However, the proteinase from Aspergillus awamori contained 16% of neutral hexoses while the other enzyme (Aspergillus niger) showed negligible amounts of these carbohydrates. Both enzymes displayed circular dichroism spectra that share a number of features with that of penicillopepsin. This suggests that proteinases from Aspergilli possess the structural folding pattern typical of aspartic proteinases. Proteolytic-activity pH optima were different, thus distinguishing one enzyme from another. This variation seems to be related to the particular resistance of the proteinases to acid denaturation, as indicated by changes in their circular dichroism spectra when the pH is decreased.

Spectrochemical evidence for the presence of a tyrosine residue in the allosteric site of glucosamine-6-phosphate deaminase from Escherichia coli.

The interaction of the enzyme glucosamine 6-phosphate deaminase from Escherichia coli with its allosteric activator, N-acetyl-D-glucosamine 6-phosphate, was studied by different spectrophotometric methods. Analysis of the circular-dichroism differential spectra produced by the binding of the allosteric activator or the competitive inhibitor 2-amino-2-deoxy-D-glucitol 6-phosphate (a homotropic ligand displacing the allosteric equilibrium to the R conformer), strongly suggests the presence of tyrosine residues at or near the allosteric site, although a conformational effect cannot be ruled out. The involvement of a single tyrosine residue in the N-acetyl-D-glucosamine-6-phosphate binding site of glucosamine-6-phosphate deaminase was supported by spectrophotometric pH titrations performed in the presence or absence of the homotropic and heterotropic ligand. In these experiments, a single titrated tyrosine residue is completely protected by saturation with the allosteric activator; this group is considerably acidic (pK 8.75). The analysis of the amino acid sequence of the deaminase using a set of indices for the prediction of surface accessibility of amino acid residues, suggests that the involved residue may be Tyr121 or Tyr254.