G. Bottura

Interaction of biotin and biotinyl derivatives with avidin: conformational changes upon binding

We used FT-Raman and FT-IR spectroscopy to study avidin and its complexes with some biotin and biotinyl derivatives where the COOH group of the pentanoic acid chain is substituted by an ester or amidic group. To investigate the small structural changes due to the binding, the amide I Raman bands of Avidin and its complexes were analysed by two fairly comparable methods. The vibrational results indicate that the interaction with the ligands slightly modifies the secondary structure of the protein, decreasing the β-sheet content and increasing the α-helix percentage. To confirm the results obtained by Raman spectroscopy, we performed Gaussian curve-fitting procedure of the amide I and III infra-red regions of neat avidin and its complexes. The percentages obtained from the analysis of the Raman and IR spectra show the same trend. Thus, IR spectroscopy as an alternative or adjunct to Raman spectroscopy is a useful tool in evaluating small structural modifications produced by a bound ligand on a protein. The structural changes are mainly due to modifications in the hydrogen bonds with the pentanoic acid moiety of the ligand and, probably, are ascribed to conformational modifications in the surface loop which binds the strands of the β-barrel. In fact, biotin-N-succinimidyl ester, as well as biotin hydrazide, which have the same ability in the hydrogen-bond formation compared with biotin, induces avidine conformational changes similar to those observed in the avidin–biotin complex. The terminal group of the biotin derivatives seems to have a slight influence since the structural modifications of the protein observed upon the binding of the long-chain hydrazide-derivative ligands (biotinyl-aminocaproic hydrazide and biocitin hydrazide) are slightly greater than those obtained with the corresponding COOH containing ligands (biotinyl-aminocaproic acid and biocytin). Although the hydrogen bonds formed by the pentanoic acid chain of biotin are not essential to complex formation since they are not directly involved in the active site, the present study confirms that they can interact with the loops that lock biotin in the β-barrels of avidin and modulate the strength of the protein-ligand interaction. Copyright

Effect of transition metal binding on the tautomeric equilibrium of the carnosine imidazolic ring

Abstract A Raman study of carnosine (Carn) and its complexes with Cu(II), Zn(II) and Co(II) at different pH values was carried out. At pH 7 and 9, Carn exists in equilibrium between two tautomeric forms. Raman spectroscopy appears to be a useful tool for analysing the tautomeric equilibrium of the imidazole ring of Carn since the sites involved in metal chelation can be identified by some bands (e.g. ν C4C5) that change in wavenumber depending on whether the imidazole ring takes the tautomeric form I or II. Form I (N π –H) is predominant in the free ligand, but the metal coordination can affect the tautomeric equilibrium. Although weak compared to those of aromatic residues, the Raman marker bands may be useful in analysing metal–histidine interaction in proteins.

Vibrational spectroscopic characterization of new calcium phosphate bioactive coatings

In this work calcium phosphate (CaP) compounds with different PO(3-)(4)/HPO(2-)(4) R molar ratios in the 0.65-149 range were synthesized. In fact, all these CaPs contain different amounts of HPO(2-)(4) and PO(3-)(4) ions as well as the amorphous precursors (tricalcium phosphate and octacalcium phosphate) of hydroxyapatite deposition, which was shown by in vitro and in vivo measurements. Spectroscopical IR and Raman results showed the presence of bands whose intensity ratio can be related to the molar ratio R; in particular, the Raman I(962)/I(987) and the IR I(1035)/I(1125) intensity ratios were characterized as markers of the molar ratio. For these CaP compounds a nucleation model, which was based on the ability of HPO(2-)(4) ions to form strong H bonds with PO(3-)(4) ions, was proposed.

Raman and IR spectra of Ca x+1.5(6-x) (HPO 4 ) x (PO 4 ) 6-x calcium phosphates with different PO 4 3- /HPO 4 2- molar ratios

Calcium phosphates with different / molar ratios have been described in the literature: octacalcium phosphate OCP, Ca8(HPO4)2(PO4)4 [1] and tricalcium phoshate TCP, Ca3(HP04)0.2(P04)1.87 [2] with / molar ratios of 2 and 9.35 respectively. These calcium phosphates are gaining increasing interest and importance because they are precursors of the formation of hydroxyapatite (HAP); therefore they can be used as coatings for metal prostheses in orthopaedics and odontostomatology.

Vibrational spectroscopy in the monitoring of chilling injury in fruits and vegetables

Vegetable marrows (cv. Seme Bolognese) and peach fruits (cv. Suncrest) were stored at different chilling temperatures in order to evaluate, by vibrational spectroscopy, the unsaturation degree of the total lipidic component and other possible markers of chilling injuries. Capillary Gas Chromatography also has been applied to evaluate the unsaturation degree of the esterified fatty acids. Both methodologies indicate a general increase of the unsaturation degrees with storage time. This can be interpreted as a better adaptation capability of the fruits to low temperatures. Moreover, the FTIR-ATR methodology points out the onset of a hydrolysis reaction of the esteric phosphate group of phospholipids during storage.