A novel fluorimetric assay for visualization and quantification of protein carbonyls in muscle foods.

Muscle foods, particularly fish products are highly exposed to oxidative stress during processing and storage, resulting in oxidative modification of proteins. Protein carbonyls content has been used as one of the measures of oxidative stress. Generally, the resulting carbonylated proteins (CPs) have so far been labeled with 2,4-dinitrophenyl (DNP) hydrazine and detected with anti-DNP antibody. However, the applicability of this method to food samples is limited by its high price, time-consuming procedure and possibility to perform the measurements just on soluble protein fractions. We developed a simpler, faster and cheaper method to assess CP level in muscle foods, including both soluble and insoluble protein fractions, which is based on a direct reaction of protein carbonyls with 7-(diethylamino)coumarin-3-carbohydrazide (CHH). The paper describes a novel technique to label both soluble and insoluble carbonylated proteins with CHH and determine carbonyl content by fluorescence microscopy assay which correlates (R = 0.911) with conventional ELISA method.

Comparison of protein oxidation and aldehyde formation during oxidative stress in isolated mitochondria.

Oxidative stress is known to cause oxidative protein modification and the generation of reactive aldehydes derived from lipid peroxidation. Extent and kinetics of both processes were investigated during oxidative damage of isolated rat liver mitochondria treated with iron/ascorbate. The monofunctional aldehydes 4-hydroxynonenal (4-HNE), n-hexanal, n-pentanal, n-nonanal, n-heptanal, 2-octenal, 4-hydroxydecenal as well as thiobarbituric acid reactive substances (TBARS) were detected. The kinetics of aldehyde generation showed a lag-phase preceding an exponential increase. In contrast, oxidative protein modification, assessed as 2,4-dinitrophenylhydrazine (DNPH) reactive protein-bound carbonyls, continuously increased without detectable lag-phase. Western blot analysis confirmed these findings but did not allow the identification of individual proteins preferentially oxidized. Protein modification by 4-HNE, determined by immunoblotting, was in parallel to the formation of this aldehyde determined by HPLC. These results suggest that protein oxidation occurs during the time of functional decline of mitochondria, i.e. in the lag-phase of lipid peroxidation. This protein modification seems not to be caused by 4-HNE.

Phenylhydrazine is a mitogen and activator of lymphoid cells.

Rats were administered a single injection of phenylhydrazine (PHZ) which induced an hemolytic anemia that reached maximal levels two to four days following injection. This was accompanied by a leukocytosis which was most pronounced four to six days after injection; lymphocytes and monocytes accounted for 75 percent to 80 percent of the leukocyte count, respectively. All peripheral blood cell values, including the red cell count and hematocrit, returned to their pre-injection levels by the 11th post-injection day. Analysis by flow cytometry of peripheral blood mononuclear cells (PBMC) isolated from PHZ-treated rats by Ficoll-Hypaque gradient separation showed a marked increase in the B cell population of the peripheral blood. This was also seen in cultures of PBMC obtained from untreated rats following incubation with PHZ. Cultures of PBMC obtained from rats four to five days after PHZ injection which were incubated with pokewood mitogen (PWM) or phytohemagglutinin (PHA) showed significant increases in blastogenesis as indicated by [3H] thymidine incorporation when compared to cultures of PBMC obtained from untreated rats incubated with these mitogens. Incubation of cultures of PBMC obtained from untreated rats with PHZ significantly increased blastogenesis in cultures of five day duration. Atypical and blastic lymphoid cells were evident in cytosmears of PBMC isolated from PHZ-treated rats and also in sections of PBMC pellets studied using the transmission electron microscope. Serum of the PHZ-treated rats contained elevated immunoglobulin titers as measured by radial immunodiffusion. The results show that PHZ stimulates lymphoid cell blastogenesis and can sensitize circulating lymphoid cells to PHA and PWM indicating that PHZ is capable of stimulating the immune system of the rat.

Temperature and pH dependence of immunoglobulin G conformation.

Previous indirect observations have indicated that IgG may change its conformation at low or high pH and at a temperature of about 35 degrees C. By means of small angle neutron scattering a change in the value of the gyration radius of two different native IgG's was observed above 44 degrees C. No similar change was detected when the sample was previously dissolved in an acidic buffer. The acidic pretreatment caused a significant decrease in the gyration radius (Rg) value measured at 20 degrees C which was partially recovered by increasing the temperature. These observations led to the assumption that the main conformational change observed appears either in the hinge region of the molecular or in the interdomain areas separating the constant and the variable domains of the Fab parts.