Protein modifications in cooked pork products investigated by a proteomic approach.

To evaluate process-induced protein modifications in cooked ham and emulsion sausages, the proteomes of whole-cut (Parma and "Praga" cooked hams) and comminuted pork (mortadella and würstel) products were compared to raw pork using two-dimensional gel electrophoresis (2-DE) coupled to image analysis and mass spectrometry (MS). Other than heat-induced breakdown of part of the myosin heavy chains, the 2-DE pattern of cooked ham was substantially similar to that of raw pork. However, the MS-based analysis showed minor modifications, including the extensive oxidation of methionines. In contrast, likely due to emulsification, comminuted sausages were characterized by an abundant insoluble protein fraction (IPF). Interestingly, tropomyosin and myosin light chains in comminuted sausages were exclusively found in the IPF. Our results indicate that the protein aggregation systems of cooked hams and emulsion sausages reflect the processing conditions and are definitely different, the former being characterized mainly by disulphide bridges and the latter by additional covalent inter-protein links.

Ferricyanide-mediated oxidation of ferrous nitrosylated sperm whale myoglobin involves the formation of the ferric nitrosylated intermediate.

Ferricyanide-mediated oxidation of ferrous oxygenated and carbonylated myoglobin (Mb(II)-O(2) and Mb(II)-CO, respectively) is limited by O(2) and CO dissociation, respectively, then the transient deoxygenated derivative (Mb(II)) is rapidly oxidized. Here, kinetics of ferricyanide-mediated oxidation of ferrous nitrosylated sperm whale myoblobin (Mb(II)-NO) is reported. Unlike for Mb(II)-O(2) and Mb(II)-CO, ferricyanide reacts with Mb(II)-NO forming first a transient ferric nitrosylated species (Mb(III)-NO), followed by the ()NO dissociation from Mb(III)-NO. Values of the second-order rate constant for ferricyanide-mediated oxidation of Mb(II)-NO (i.e., for the formation of the transient Mb(III)-NO species) and of the first-order rate constant for ()NO dissociation from Mb(III)-NO (i.e., for Mb(III) formation) are (1.3+/-0.2)x10(6)M(-1)s(-1) and 7.6+/-1.3s(-1), respectively, at pH 8.3 and 20.0 degrees C. Since ()NO dissociation from Mb(II)-NO is very slow, and (unlike O(2) and CO) ()NO is a ligand for both Mb(II) and Mb(III), Mb(II)-NO can be oxidized without requiring ()NO dissociation.

A comparative study on axial coordination and ligand binding in ferric mini myoglobin and horse heart myoglobin.

The absorption and resonance Raman spectra and the azide binding kinetics of ferric horse heart myoglobin (Mb) and mini myoglobin (a chemically truncated form of horse heart Mb containing residues 32-139) have been compared. The steady-state spectra show that an additional six-coordinated low-spin form (not present in entire horse heart Mb, which is purely six-coordinated high spin) predominates in mini Mb. The distal histidine is possibly the sixth ligand in this species. The presence of two species corresponds to a kinetic biphasicity for mini Mb that is not observed for horse heart Mb. Azide binds to horse heart Mb much more slowly than to sperm whale Mb. This difference may result from a sterically hindered distal pocket in horse heart Mb. In both cases, the rate constants level off at high azide concentrations, implying the existence of a rate-limiting step (likely referable to the dissociation of the axial sixth ligand). The faster rate constant of mini Mb is similar to that of sperm whale Mb, whereas the slower one is similar to that of entire horse heart Mb.

rhEPO (recombinant human eosinophil peroxidase): expression in Pichia pastoris and biochemical characterization.

A Pichia pastoris expression system has for the first time been successfully developed to produce rhEPO (recombinant human eosinophil peroxidase). The full-length rhEPO coding sequence was cloned into the pPIC9 vector in frame with the yeast alpha-Factor secretion signal under the transcriptional control of the AOX (acyl-CoA oxidase) promoter, and transformed into P. pastoris strain GS115. Evidence for the production of rhEPO by P. pastoris as a glycosylated dimer precursor of approx. 80 kDa was determined by SDS/PAGE and gel filtration chromatography. Recombinant hEPO undergoes proteolytic processing, similar to that in the native host, to generate two chains of approx. 50 and 20 kDa. A preliminary biochemical characterization of purified rhEPO demonstrated that the spectral and kinetic properties of the recombinant wild-type EPO are comparable with those of the native enzyme and are accompanied by oxidizing activity towards several physiological anionic substrates such as SCN-, Br- and Cl-. On the basis of the estimated K(m) and kcat values it is evident that the pseudohalide SCN- is the most specific substrate for rhEPO, consistent with the catalytic properties of other mammalian EPOs purified from blood.