Cytochrome c peroxidase (CCP) catalyses the reduction of H2O2 to H2O, an important step in the cellular detoxification process. The crystal structure of the di-heme CCP from Pseudomonas nautica 617 was obtained in two different conformations in a redox state with the electron transfer heme reduced. Form IN, obtained at pH 4.0, does not contain Ca2+ and was refined at 2.2 Angstrom resolution. This inactive form presents a closed conformation where the peroxidatic heme adopts a six-ligand coordination, hindering the peroxidatic reaction from taking place. Form OUT is Ca2+ dependent and was crystallized at pH 5.3 and refined at 2.4 Angstrom resolution. This active form shows an open conformation, with release of the distal histidine (His71) ligand, providing peroxide access to the active site. This is the first time that the active and inactive states are reported for a di-heme peroxidase.

This study describes the characterisation of whey protein hydrolysates obtained from tryptic hydrolysis to assess their application as ingredients with angiotensin-converting-enzyme (ACE) inhibitory action. The levels of a-lactalbumin (alpha-la) and P-lactoglobulin (beta-lg) remaining after hydrolysis were quantified. Peptides were separated by RP-HPLC, and Ala-Leu-Pro-Met-His-Ile-Arg (ALPMHIR), the most potent beta-lg-derived ACE-inhibitory peptide was monitored. A correlation curve was established for the production of this peptide as a function of hydrolysis time. Heat-induced gelation of hydrolysates was studied by small-deformation rheology. The gelation times and the strength of the final gels were highly dependent on the degree of hydrolysis. Smaller peptides liberated by hydrolysis contributed to the inability of whey protein hydrolysates to gel. (c) 2006 Elsevier Ltd. All rights reserved.

A [2Fe-2S] cluster has been detected in mammalian ferrochelatase, the terminal enzyme of the heme biosynthetic pathway. Natural ferrochelatase, purified from mouse livers, and recombinant ferrochelatase, purified from an overproducing strain of Escherichia coli, were investigated by electron paramagnetic resonance (EPR) and Mossbauer spectroscopy. In their reduced forms, both the natural and recombinant ferrochelatases exhibited an identical EPR signal with g values (g = 2.00, 1.93, and 1.90) and relaxation properties typical of [2Fe-2S](+) cluster. Mossbauer spectra of the recombinant ferrochelatase, purified from a strain of E. coli cells transformed with a plasmid encoding murine liver ferrochelatase and grown in Fe-57-enriched medium, demonstrated unambiguously that the cluster is a [2Fe-2S] cluster. No change in the cluster oxidation state was observed during catalysis, The putative protein binding site for the Fe-S cluster in mammalian ferrochelatases is absent from the sequences of the bacterial and yeast enzymes, suggesting a possible role of the [2Fe-2S] center in regulation of mammalian ferrochelatases.

The simultaneous separation and quantification of two casein peptides (IPP, VPP) presenting potent inhibitory activity of angiotensin-converting-enzyme (ACE) and casein in fermented milks was developed. Gradient elution was carried out at a flow-rate of 1 mL/min, using a mixture of two solvents. Solvent A was 0.1% TFA in water and solvent B was acetonitrile-water-trifluoracetic acid 95:5:0.1. The effluent was monitored by UV detector at 214 nm. Calibration curves were constructed in the interval of 0.01-1.0 mg/mL for VPP, 0.005-1.0 mg/mL for IPP, and 0.05-3.0 mg/mL for casein. R 2 invariably exceeded 0.999. The detection limits were 0.004 for VPP, 0.002 mg/mL for IPP, and 0.02 mg/mL for casein. Repeatability of the method was evaluated by six consecutive injections of two standard solutions containing VPP, IPP, and casein. The RSD values for concentration were all below 5.08%. Recovery studies were carried out to determine the accuracy of the method. Recoveries ranged between 88 and 98.2%. The methodology was applied, not only, for the monitorization of VPP, IPP, and casein in commercial fermented milks labeled as presenting anti hypertensive properties, but also, in milk with different degrees of fermentation by L Helveticus, and in other commercial functional fermented milks, such as, those presenting cholesterol lowering properties.