Several studies have shown that anions induce collapse of acid-denatured cytochrome c into the compact A state having the properties of the molten globule and that the anion charge is the main determinant for the A state stabilization. The results here reported show that the anion size plays a role in determining the overall structure of the A state. In particular, small anions induce formation of an A state in which the native Met80-Fe(III) axial bond is recovered and the nativelike redox properties restored. On the other hand, the A state stabilized by large anions shows a histidine (His26 or His33) as the sixth ligand of the heme-iron, a very weak interaction between Trp59 and the heme propionate, and lacks nativelike redox properties. The two anion-stabilized states show similar stability, indicating that (i) the hydrophobic core (which is equally stabilized by all the anions investigated, independently of their size) is the region that mainly contributes to the macromolecule stabilization, and (ii) the flexible loops are responsible for the spectroscopic (and, thus, structural) and redox differences observed.

The performances and the stability of a novel subcutaneous glucose monitoring system have been evaluated. GlucoDay † (A. Menarini I.F.R. S.r.l, Florence Italy) is a portable instrument provided with a micro-pump and a biosensor coupled to a microdialysis system capable of recording the subcutaneous glucose level every 3 min. Long and short term stability of the biosensor are discussed and the results of some critical in vitro and in vivo (on rabbits) experiments are reported. A linear response up to 30 mM has been found for in vivo glucose concentration. The sensitivity referred to blood glucose is better than 0.1 mM and the zero current is typically below the equivalent of 0.1 mM. In the accuracy study a mean bias of 2.7 mg/dl and a correlation coefficient equal to 0.9697 have been found. At room temperature, an excellent membrane stability assures good performances up to 6 months from the first use. #

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Redox potentials of recombinant manganese peroxidase from Phanerochaetes chrysosporium have been measured by cyclic voltammetry as a function of pH, between pH 4.5 and pH 10.5. They display a bimodal behaviour (characterized by an 'alkaline' and an 'acid' transition), which indicates that (at least) two protonating groups change their pK(b) values upon reduction (and/or oxidation) of the iron atom in haem. Analogous measurements have been carried out on four site-directed mutants involving residues in close proximity to the proximal ligand, His(173), in order to investigate the role played by residues of the proximal haem pocket on the redox properties of this enzyme. Results obtained suggest that the protonation state of N(delta) of the proximal imidazole group is redox-linked and that it is crucial in regulating the 'alkaline' transition. On the other hand, none of the proximal mutants alters the 'acid' transition, suggesting that it is modulated by groups located in a different portion of the protein.

Redox potentials of recombinant manganese peroxidase from Phanerochaetes chrysosporium have been measured by cyclic voltammetry as a function of pH, between pH 4.5 and pH 10.5. They display a bimodal behaviour (characterized by an ‘alkaline’ and an ‘acid’ transition), which indicates that (at least) two protonating groups change their pKb values upon reduction (and/or oxidation) of the iron atom in haem. Analogous measurements have been carried out on four site-directed mutants involving residues in close proximity to the proximal ligand, His173, in order to investigate the role played by residues of the proximal haem pocket on the redox properties of this enzyme. Results obtained suggest that the protonation state of N∆ of the proximal imidazole group is redox-linked and that it is crucial in regulating the ‘alkaline’ transition. On the other hand, none of the proximal mutants alters the ‘acid’ transition, suggesting that it is modulated by groups located in a different portion of the protein.

At high concentration (98% or higher, v/v), glycerol induces collapse of acid-denatured cytochrome c into a compact state, the G(U) state, showing a molten globule character. The G(U) state possesses a nativelike alpha-helix structure but a tertiary conformation less packed with respect to the native state. The spectroscopic properties of the G(U) state closely resemble those of the molten globule stabilized by the organic solvent from the native protein (called the G(N) state), indicating that glycerol can stabilize the molten globule of cytochrome c either from the native or the acid-denatured protein. The G(U) and the G(N) states show spectroscopic (and, thus, structural) properties and stabilities comparable to those of molten globules stabilized by different effectors, despite the fact that the mechanisms involved in the molten globule formation may significantly differ. This implies in cytochrome c a hierarchy for the rupture (native-to-molten globule) or the formation (unfolded-to-molten globule) of intramolecular interactions leading to the stabilization of the molten globule state of the protein, independently from the effector responsible for the structural transition, in accord with the sequential model proposed by Englander and collaborators.