Ciro Balestrieri

Simultaneous determination of tyrosine and tryptophan residues in proteins by second-derivative spectroscopy

Abstract The mutual interference between the second-derivative bands of tyrosine and tryptophan in proteins has been evaluated in terms of the ratio r between two peak-to-peak distances. The r values have been found to be well related, although not linearly, to the tyrosine/tryptophan ratio in both model compound mixtures and proteins. A method for the simultaneous determination of the two major protein chromophores at neutral pH has been developed.

Pectin methyl esterase from Actinidia chinensis fruits

Abstract Pectin methylesterase (EC 3.1.1.11) from Actinidia chinensis fruit, cv Hayward, consists of two forms (PME1 and PME2) separable by heparin-Sepharose chromatography. The two forms have been purified to homogeneity to respective specific activities of 933 and 974 units mg−1. The most efficient step in the purification procedure was the affinity chromatography on heparin-Sepharose column. The enzymatic preparations exhibit the same Mr in native as well as under denaturing conditions; a value of 57,000 was found in ultracentrifugal experiments. The two forms possess the same isoelectric point of 7.3, but differ in their affinity toward citrus pectin having a Km of 1.82 mg ml−1 for PME1 and 0.76 mg ml−1 for PME2. They also differ in thermostability, where PME1 is more stable than PME2. The two forms are glycoproteins with a different degreeof glycosylation as shown by different retention time on concanavalin A-Sepharose with respect to a glucose gradient.

Second-derivative spectroscopy of proteins: Studies on tyrosyl residues

Ionization of the phenolic group of N-acetyltyrosynamide has been studied using second-derivative spectroscopy. At pH 12.5 the second-derivative spectrum of the model compound revealed the presence of derivative bands in a spectral region (between 250 and 270 nm) where interference coming from other ultraviolet-absorbing chromophores is negligible. One of these peaks (260-nm peak) has been employed for the determination of tyrosyl groups in mixtures containing the aromatic amino acids.

Cellulose-based hydrogels as body water retainers

In this work the possibility of using hydrogels as body water retainers for a therapeutic aid in pathologies such as oedemas of various origins was explored. For such a purpose, the material requires a good compatibility and a controlled swelling capacity without altering the body electrolyte homeostasis. The hydrogel was designed to meet the swelling requirements with the physiological constraints and its biocompatibility was assessed either in vitro or in vivo. Absorption tests were performed in order to define the swelling behavior by varying the pH and ion content of the external solution. The hydrogel swelling capacity was assessed in the presence of various solvents, in order to evaluate its absorption capacity in solutions similar to biological fluids. In addition, the capacity of the gel to modify electrolyte homeostasis by adsorbing ions such as calcium, potassium and sodium was tested. In order to assess the gel biocompatibility after contact of the hydrogel with intestinal cells, arachidonic acid relase was determined. No significant intracellular increase of free arachidonic acid was found in the cells after up to 2 h of contact with the gel. The results suggest that, as far as brief periods are concerned, the gel does not cause an inflammatory response in intestinal cells. ©2000 Kluwer Academic Publishers

Equilibrium evidence of non-single step transition during guanidine unfolding of apomyoglobins

There are many experimental evidences that all myoglobins have the same basic structure [l-4]. It is generally assumed that the denaturation of these proteins is a single step process with variations in all physical properties which are closely related in occurrence and extent -[3,5,6]. In this paper we report the effect of guanidine hydrochloride on intrinsic fluorescence and that of the I-anilino&naphthalene sulfonate (ANS) conjugate of four different apomyoglobins, i.e. tuna, buffalo, beef, and sperm whale. Tryptophan residues are located in the N-terminal branch of the globin molecule [7 3 , whereas ANS is known to bind the apoprotein in the same non-polar moiety of the heme [8]. The N-terminal region is not involved in the formation of the heme pocket [9], therefore the environmental changes in the two molecular districts of the examined globins are independently evidenced by the variation of fluorescence behaviour observed in the two chromophores. The experimental data show an unexpected difference in sperm whale and tuna globin unfolding. The results are indicative that, during the course of denaturation, the conformational changes in the tryptophanyl segment are not thoroughly concomitant with those involving the heme pocket.

New unusual pregnane glycosides with antiproliferative activity from Solenostemma argel

Seven new 15-keto pregnane glycosides, namely Stemmosides E--K, were isolated from Solenostemma argel. Stemmosides E--J are characterized by the occurrence of an uncommon 14 beta proton configuration while stemmosides E and F possess in addition a rare enolic function in C-16. On the other hand, stemmosides G-J display an unusual C-17 alpha side chain. Their structures were established by ESI-MS and NMR experiments. Moreover, the effect of these compounds on the VEGF-induced in Kaposis sarcoma cell proliferation was tested. Results indicated that all the compounds reduced the cell proliferation in a dose dependent manner.

The effect of evolution on homologous proteins: A comparison between the chromophore microenvironments of italian water buffalo (Bos bubalus, L.) and sperm whale apomyoglobin

The perturbing effect of guanidium hydrochloride and pH on the molecular structure of water buffalo apomyoglobin has been investigated by circular dichroism in the far and near ultraviolet and by fluorescence. In the wavelength region between 320 and 260 nm the circular dichroic spectrum of the globin is highly structured and the contributions of the aromatic chromophores have been resolved. Buffalo apomyoglobin undergoes a structural transition at neutral pH which involves elements of the secondary and tertiary structure, as indicated by changes of dichroic activity of the peptide and aromatic chromophores and the fluorescence of the two tryptophanyl residues. The possibility of charge-transfer complex between indole and imidazole is discussed. A major structural transition with abrupt unfolding takes place in the pH region between 5.6 and 4.3. Below pH 4.3 the peptide helical residues, which survive the acid transition, appear to be resistent to further acidification to pH 2.0 while tryptophanyl emission is quenched and shifted to longer wavelengths. A structural transition occurs also in alkali above pH 10, which has been detected by the same techniques. The relationships between buffalo and sperm whale apomyoglobin are discussed.

Structural and functional aspects of the heart ventricle myoglobin of bluefin tuna

The heart ventricle myoglobin of bluefin tuna has been purified to an apparent homogeneity. The amino acid analysis has revealed only a limited number of substitutions between the myoglobins of yellowfin and bluefin tuna. The alpha-helix content of tuna myoglobin has been found considerably lower than that of mammalian myoglobin. No correlation has been discovered between the conformational stability and alpha-helix content. Denaturation experiments have shown that the whole structure of tuna myoglobin results from the interaction of two structural units which represent the product of independent folding processes. The structure of tuna myoglobin has been found more open and disorganized than that of sperm whale. This result has been related to the low content of electrostatic interactions and explained in terms of evolutive adaptations.

Amino acid composition and physico-chemical properties of bluefin tuna (Thunnus thynnus) myoglobin.

1. The heart ventricle myoglobin of Atlantic bluefin tuna has been purified and its amino acid composition has been determined. 2. The perturbing effect of guanidine hydrochloride on the molecular structure of tuna ferrimyoglobin and its corresponding apoprotein has been investigated by Soret absorbance and ultraviolet fluorescence. 3. The conformation-free energy of unfolding delta G0 has been calculated by thermodynamic treatments of the data concerning guanidine unfolding. 4. The results have been compared with other known myoglobins, particularly those of yellowfin tuna.

Simultaneous determination of lysophospholipids by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatography (HPLC) procedure for the separation of choline lysophospholipids including 1-acyl-lysophosphatidylcholines and 1-O-alkyl-lysophosphatidyl- cholines, like the lysoform of the platelet activating factor (2-lysoPAF), is described. The lysophospholipids are derivatized at the sn-2 position of the hydroxyl group by 7-diethylaminocoumarin-3-carbonylazide, which converts them into the corresponding carbamoyl derivatives. The derivatized compounds were well separated by reversed-phase HPLC and quantified by fluorimetric detection. This method shows a high sensitivity and allows the separation and quantification of mixtures of lysophospholipids at picomolar level. The method was applied to assay enzyme activities, like phospholipase A2 and PAF-acetylhydrolase, on single phospholipids or their mixtures.