Carlo Fini

Porcine odorant-binding protein: structural stability and ligand affinities measured by Fourier-transform infrared spectroscopy and fluorescence spectroscopy

Infrared spectra show that the binding of the odorants 2-isobuthyl-3-methoxypyrazine (PYR) and 3,7-dimethyl-1-octanol (DMO) stabilises the tertiary structure of porcine OBP-I against thermal denaturation. The fluorescence emission spectrum of the single tryptophan shows a lambdamax at 337 nm, indicating that the residue is not directly exposed to the solvent. Tryptophan does not appear to be involved in the odorant binding process and it is not accessible to the fluorescence quenchers NaI, CsCl and acrylamide. The binding of the fluorescent dye 1-aminoanthracene (1-AMA), a strong ligand, does not modify the tryptophan fluorescence spectrum. In contrast, the lambdamax of 1-AMA bound to OBP-I is shifted from 537 to 481 nm, with a lambdamax intensity increase by a factor of 80. Bound 1-AMA is displaced by odorant molecules in competitive binding assays and can be employed in simple and rapid binding assay, avoiding the use of radioactive ligands. The Scatchard plot shows that 1-AMA binds to OBP-I with a dissociation constant of 1.3 microM and an equimolar stoichiometry.

Simultaneous analysis of bases, nucleosides and nucleoside mono- and polyphosphates by high-performance liquid chromatography

A single-column high-performance liquid chromatographic system for the simultaneous separation of bases, nucleosides and nucleoside mono- and polyphosphates has been developed, in which a strong porous anion-exchange resin (Aminex A-14) is used. The chromatographic run, carried out at 55 degrees and at alkaline pH by using a linear gradient both of ionic strength and pH, takes less than 225 min. The quantitative application of the described procedure to the analysis of cell nucleotide pools is reported.

High-performance liquid chromatographic analysis of free hydroxyproline and proline in blood plasma and of free and peptide-bound hydroxyproline in urine.

A rapid, accurate and sensitive method for the determination of free hydroxyproline and proline in plasma and of total hydroxyproline in urine has been developed. Free imino acids and internal standard are extracted from plasma by trichloroacetic acid precipitation of protein and they are selectively derivatized with 4-chloro-7-nitrobenzofurazan, after reaction of the acid extract with o-phthalaldehyde. The highly fluorescent adducts of imino acids are separated on a Spherisorb ODS 2 reversed-phase column using acetonitrile-0.1 M sodium phosphate buffer, pH 7.2 (9:91, v/v) as mobile phase, followed by fluorometric detection. Total hydroxyproline determination in urine hydrolysates is carried out by reaction of the imino acid with 4-chloro-7-nitrobenzofurazan after clean-up on a Sep-Pak C18 cartridge of the o-phthalaldehyde-treated sample, high-performance liquid chromatographic separation and fluorometric quantitation of the derivative.

Hydrophobic interactions and ionic networks play an important role in thermal stability and denaturation mechanism of the porcine odorant-binding protein.

Despite the fact that the porcine odorant‐binding protein (pOBP) possesses a single tryptophan residue (Trp 16) that is characterized by a high density microenvironment (80 atoms in a sphere with radius 7 Å) with only one polar group (Lys 120) and three bound water molecules, pOBP displayed a red shifted fluorescence emission spectrum (λmax = 340 nm). The protein unfolding in 5M GdnHCl was accompanied by the red shift of the fluorescence emission spectrum (λmax = 353 nm), by the increase of fluorescence quantum yield, and by the decrease of lifetime of the excited state (from 4.25 ns in native state to 3.15 ns in the presence of 5M GdnHCl). Taken together these data indicate the existence of an exciplex complex (Trp 16 with Lys 120 and/or with bound molecules of water) in the protein native state. Heat‐induced denaturation of pOBP resulted in significant red shifts of the fluorescence emission spectra: the value of the ratio (I320/I365) upon excitation at λex = 297 nm (parameter A) decreases from 1.07 to 0.64 passing from 60 to 85°C, and the calculated midpoint of transition was centered at 70°C. Interestingly, even at higher temperature, the values of the parameter A both in the absence and in the presence of GdnHCl did not coincide. This suggests that a portion of the protein structure is still preserved upon the temperature‐induced denaturation of the protein in the absence of GdnHCl. CD experiments performed on pOBP in the absence and in the presence of GdnHCl and at different temperatures were in agreement with the fluorescence results. In addition, the obtained experimental data were corroborated by the analysis of the 3D structure of pOBP which revealed the amino acid residues that contribute to the protein dynamics and stability. Finally, molecular dynamics simulation experiments pointed out the important role of ion pair interactions as well as the molecular motifs that are responsible for the high thermal stability of pOBP, and elucidated the reasons of the protein aggregation that occurred at high temperature. Proteins 2008.

Purification, cloning and characterisation of odorant- and pheromone-binding proteins from pig nasal epithelium.

Abstract. Two distinct classes of lipocalin isoforms (OBP-IIs and OBP-IIIs) were purified and identified from porcine nasal mucosa of male and female individuals. Using primers designed on their N-terminal sequence, the complete primary structures of the mature polypeptides were determined. Mass spectrometry analysis confirmed the identity of the cDNA-derived sequences and provided information regarding their post-translational modifications. These species strongly resemble a lipocalin expressed by von Ebners gland and salivary lipocalins carrying sex-specific pheromones secreted only by the boars submaxillary glands. Both OBP-IIs and OBP-IIIs present two cysteines paired in a disulphide bond; the remaining residues occur in a reduced form. In addition, OBP-IIIs are heavily glycosylated and markedly different in their glycan moiety from the salivary lipocalins. A three-dimensional model is proposed based on protein species with known structure. Like salivary lipocalins, OBP-IIIs bind a number of odorant molecules, with highest affinity for the specific pheromone 5α-androst-16-en-3-one. The high similarity between OBPs from the nasal area and lipocalins from secretory glands suggests a common function in binding the same pheromonal ligands, the latter carrying chemical messages into the environment the former delivering them to specific receptors.

Thermodynamics and kinetics of the thermal unfolding of plastocyanin

Abstract The thermal denaturation of plastocyanin in aqueous solution was investigated by means of DSC, ESR and absorbance techniques, with the aim of determining the thermodynamic stability of the protein and of characterizing the thermally induced conformational changes of its active site. The DSC and absorbance experiments indicated an irreversible and kinetically controlled denaturation path. The extrapolation of the heat capacity and optical data at infinite scan rate made it possible to calculate the kinetic and thermodynamic parameters associated with the denaturation steps. The denaturation pathway proposed, and the parameters found from the calorimetric data, were checked by computer simulation using an equation containing the information necessary to describe the denaturation process in detail. ESR and absorbance measurements have shown that structural changes of the copper environment occur during the protein denaturation. In particular, the geometry of the copper-ligand atoms changes from being tetrahedral to square planar and the disruption of the active site precedes the global protein denaturation. The thermodynamic enthalpic change, the half-width transition temperature, and the value of ΔCp, were used to calculate the thermodynamic stability, ΔG, of the reversible process over the entire temperature range of denaturation. The low thermal stability found for plastocyanin, is discussed in connection with structural factors stabilizing the native state of a protein.

Type III procollagen peptide and PZ-peptidase serum levels in pre-cirrhotic liver diseases

To obtain a dynamic and non-invasive picture of hepatic fibrosis in pre-cirrhotic liver diseases we measured both the concentration of the N-terminal peptide of procollagen III, as a marker of collagen synthesis, and the activity of PZ-peptidase, an enzyme involved in collagen degradation, in the serum of alcoholic or chronic viral hepatitis patients. Peptide serum levels were similar in chronic persistent hepatitis and controls, but significantly higher in chronic active hepatitis. Chronic persistent hepatitis patients had PZ-peptidase levels higher than controls, but similar to chronic active hepatitis. The increase in collagen synthesis without a parallel increase in collagen degradation seen in chronic active hepatitis could be regarded as a sign of impending cirrhosis, whereas the unbalanced rise in PZ-peptidase observed in chronic persistent hepatitis is consistent with the non-progressive character of this disorder. In alcoholic hepatitis both peptide concentration and PZ-peptidase activity were elevated, thus suggesting that both collagen synthesis and degradation are activated. However, the greater increase in PZ-peptidase than in peptide serum levels seen in some patients seems to indicate a minor tendency to progressive fibrosis or a trend towards resolution. Unlike liver disease patients, normal peptide and PZ-peptidase levels were found in patients with pancreatic fibrosis. Since circulating inhibitors and activators of the PZ-peptidase activity can be excluded, as proved by this study, joint peptide and PZ-peptidase serum measurements would seem to offer a simple reliable non-invasive method for differentiating and monitoring progressive and non-progressive forms of hepatic fibrosis.

Analysis by High Performance Liquid Chromatography of The Water-Soluble Precursors of Choline and Ethanolamine Phosphoglycerides in the Rat Brain

Abstract The simultaneous quantitation of PC, PE, CDP-cho-line and CDP-ethanolamine has been performed by high performance liquid chromatography of purified rat brain extracts. The water-soluble intermediates of the pho-sphoglyceride biosynthesis were extracted from the rat brain by homogenization in 1 M PCA and purified by chro-matography on neutral alumina and Dowex 1. This procedure removes almost all of the UV-contaminating substances and inorganic phosphate, which interfere in the a-nalytical chromatographic process. The high performance liquid chromatography was carried out using the styre-ne-type Aminex A-14 resin. The elution was performed for 5 min with 0.1 M 2-amino-2-methyl-1-propanol, 0.02 M NaCl, pH 10.3 buffer, and then with 0.1 M 2-amino-2-me-thyl-1-propanol, 0.17 M NaCl, pH 11.0 solution, until the end of the run. Satisfactory separations of PC, PE, CDP-choline and CDP-ethanolamine were achieved within 30 min. 5′-CMP can also be dosed. The cytidine-containing compounds have been detected a...

High-performance liquid chromatographic method for the determination of prolyl peptides in urine

A rapid and accurate method is described for the determination of prolyl peptides in urine, with specific reference to the dipeptide prolylhydroxyproline, and free hydroxyproline and proline. Free amino acids and peptides were isolated from urine on cation-exchange minicolumns, and free imino acids and prolyl-N-terminal peptides were selectively derivatized with 4-chloro-7-nitrobenzofurazan, after reaction of amino acids and N-terminal aminoacyl peptides with o-phthalaldehyde. The highly fluorescent adducts of imino acids and prolyl peptides were separated on a Spherisorb ODS 2 column by isocratic elution for 12 min using as mobile phase 17.5 mM aqueous trifluoracetic acid solution containing 12.5% acetonitrile (eluent A), followed by gradient elution from eluent A to 40% of 17.5 mM aqueous trifluoroacetic acid solution containing 80% acetonitrile in 20 min. Analytes of interest, in particular the dipeptide prolylhydroxyproline, can be easily quantified by fluorimetric detection (epsilon ex = 470 nm, epsilon em = 530 nm) without interference from primary amino-containing compounds.

High Performance Liquid Chromatographic Analysis of S-Adenosylmethionine and S-Adenosylhomocysteine in Rat Liver

Abstract A sensitive and precise high performance liquid chromatographic method for the simultaneous quantitation of S-adenosylmethionine (SAMe) and S-adenosylhomocysteine (SAH) in rat liver is described. Liver is extracted by homogenization in 1.5 M PCA. SAMe and SAH are purified from contaminating substances by chromatography on Dowex 50 W × 2 and analyzed by HPLC on conventional styrene-type anion exchanger. Analytical chromatography, performed under i-socratic elution conditions, is achieved in 18 min. SAH is determined as such while SAMe as adenine after hydrolysis at 60°C, un der alkaline conditions. The sensitivity of the analytical proce dure allows the determination of SAMe and SAH in less than 200 mg of liver sample.