A methodology for monitoring globular milk protein changes induced by ultrafiltration: a dual structural and functional approach.

Understanding filtration mechanisms at a molecular level is important for predicting structural and functional properties of globular milk proteins after membrane operations. This stage is thus highly decisive for the further development of membrane separations as an efficient alternative to chromatographic processes for the fractionation of milk proteins. In this study, we proposed an original and complete analytical package for the examination of the putative effect of filtration at both macroscopic and molecular levels. We then investigated the pertinence of this analytical package during ultrafiltration (UF) of globular milk proteins in both dead-end and crossflow modes. Reverse-phase HPLC combined with statistical computing was shown to be relevant for the assessment of even slight physically induced modifications. Adaptations of circular dichroism and solubility measurements, regarding their respective dependence on temperature and pH, were also useful for an accurate evaluation of functional modifications. At last, immunochemistry was proven to be a pertinent tool for the specific detection of modifications affecting a targeted protein, even in mixed solutions. Moreover, results obtained by such methods were shown to be coherent with data obtained from classical techniques such as fluorescence. For beta-lactoglobulin, some physically induced modifications were noticed in the permeate because of shear stress inside membrane pores. In the case of alpha-lactalbumin dead-end UF, permeation was shown to affect protein characteristics because of an increase in the relative calcium content responsible for a conformational transition from the apo-form to the holo-form of the protein. Finally, during crossflow UF with limited transmission of BSA, observations were coherent with a partial aggregation because of the circulation of proteins in the filtration pilot. Such a hypothesis corroborates results previously mentioned in the literature.

Simple rapid procedure for preparation of large quantities of ovalbumin.

A simple rapid procedure for preparation of large quantities of highly purified homogeneous ovalbumin from egg white by using an anion exchanger is described. It is based on the principle of frontal chromatography. The volume of "mucin-free" egg white loaded onto the column was determined in order to exceed resin capacity. Thus, competition between proteins for resin sites was created. Owing to its high negative charge density, ovalbumin drives other egg white proteins from the column progressively. Two hundred and fifty milliliters of Q-Sepharose FF gel eluted isocratically with 0. 5 M NaCl extracted 9.55 g of ovalbumin with a purity rate of 83%. A 6.75 g amount of ovalbumin, with a purity rate of 94%, was recovered with an isocratic elution program using 0.14 M NaCl. Purified ovalbumins were compared by electrophoresis and analytical chromatography with other ovalbumin preparations.

Application of chromatography and mass spectrometry to the characterization of food proteins and derived peptides.

The following review describes the development of mass spectrometry off-line and on-line coupled with liquid chromatography to the analysis of food proteins. It includes the significant results recently obtained in the field of milk, egg and cereal proteins. This paper also outlines the research carried out in the area of food protein hydrolysates, which are important components in foodstuffs due to their functional properties. Liquid chromatography and mass spectrometry have been particularly used for the characterization of food peptides and especially in dairy products.

The role of Mg2+ in the hydrolytic activity of the isolated chloroplast ATPase: study by high-performance liquid chromatography.

The influences of total magnesium ion concentration at different total ATP concentrations, and of total ATP concentration, for different total magnesium ion concentrations, on the enzymatic rate of the isolated chloroplast F1 ATPase, have been followed by a chromatographic method consisting in the separation and determination of ADP. From the various series of curves, it is concluded that the experimental results (position of the maxima, Km values) are better fitted by a mechanism involving the activation of the enzyme by magnesium ion and hydrolysis of free ATP, rather than by the classical mechanism, for which the enzyme hydrolyzes the MgATP complex and is inhibited by Mg2+. Although the equations giving the reaction rate are similar in the two cases, the calculated values of Km are widely different. The value obtained from the classical mechanism does not agree with KD, the dissociation constant of the enzyme-substrate complex, measured by the Hummel and Dreyer method. Moreover, when the total ATP concentration tends toward the total magnesium ion concentration, the nucleotide binding to the enzyme tends toward zero, although it should be maximum if MgATP were the true substrate. Finally, the inhibitory effect of Na+ is more easily explained as a competition between this ion and the activating Mg2+, than by the classical mechanism.

The prokaryotic thermophilic TF1-ATPase is functionally compatible with the eukaryotic CFo-part of the chloroplast ATP-synthase.

The ATP synthase from chloroplasts, CFo.F1, was reconstituted into liposomes, from which most of CF1 was removed by a short treatment with guanidinium chloride. ATP-dependent proton uptake was restored with these CFo-liposomes even better by the addition of the bacterial TF1-than of the related CF1-part. This proton uptake was prevented by tentoxin, a specific inhibitor of the CF1-ATPase, in these CFo.F1-liposomes, but not in the hybrid CFo.TF1-liposomes. Venturicidin, a specific inhibitor of proton flow through CFo, was able to block it in both the hybrid CFo.TF1-liposomes and reconstituted CFo.F1-liposomes. These results indicate that the bacterial TF1-part binds to the eukaryotic CFo-part of four subunits forming a functional CFo.TF1-ATPase.

The Rieske FeS center from the gram-positive bacterium PS3 and its interaction with the menaquinone pool studied by EPR.

The Rieske 2Fe2S center from Bacillus PS3, a Gram-positive thermophilic eubacterium, has been studied by EPR spectroscopy. Its redox midpoint potential at pH 7.0 was determined to be +165 +/- 10 mV and was found to decrease with an apparent slope of -80 mV/pH unit above pH 7.9. The Qo-site inhibitor stigmatellin induced spectral changes analogous to those reported for Rieske centers from mitochondria and chloroplasts. The redox midpoint potential of the PS3 Rieske cluster was not affected by stigmatellin. The orientation of the g tensor was similar to other Rieske centers (gz and gy are oriented parallel, gx is oriented perpendicular to the membrane plane). The shape of the EPR spectrum of the Rieske cluster from PS3 changed as a function of the redox state of the menaquinone (MK) pool. This permitted the redox midpoint potential of the MK pool to be determined in the membrane. Values of -60 +/- 20 mV at pH 7.0 and of -130 +/- 20 mV at pH 8.0 were obtained. The results are compared with already published data from other Rieske centers. It is proposed that all Rieske centers that function in electron transport chains using MK as pool quinone show common features that distinguish them from Rieske centers operating in ubiquinone- or plastoquinone-based electron transfer chains.