Efecto de la hipoxia-reoxigenación y las radiaciones ionizantes en la captación de glucosa en líneas tumorales de seno y colon cocultivadas con células endoteliales.

La captación de glucosa y su conversión en lactato juega un papel fundamental en el metabolismo tumoral, independientemente de la concentración de oxígeno presente en el tejido (efecto Warburg). Sin embrago, dicha captación varía de un tipo tumoral a otro, y dentro del mismo tumor, situación que podría depender de las características microambientales tumorales (fluctuaciones de oxígeno, presencia de otros tipos celulares) y de factores estresores asociados a los tratamientos. Se estudió el efecto de la hipoxia-reoxigenación (HR) y las radiaciones ionizantes (RI) sobre la captación de glucosa, en cultivos de líneas tumorales MCF-7 y HT-29, cultivadas de forma aislada o en cocultivo con la línea celular EAhy296. Se encontró que la captación de glucosa en HR es diferente para lo descrito en condiciones de hipoxia permanente y que es modificada en el cocultivo. Se identificaron poblaciones celulares dentro de la misma línea celular, de alta y baja captación de glucosa, lo que implicaría una simbiosis metabólica de la célula como respuesta adaptativa a las condiciones tumorales. Se evaluó la expresión de NRF2 y la translocación nuclear de NRF2 y HIF1a, como vías de respuesta a estrés celular e hipoxia. La translocación nuclear de las proteínas evaluadas explicaría el comportamiento metabólico de las células tumorales de seno, pero no de colon, por lo cual deben existir otras vías metabólicas implicadas. Las diferencias en el comportamiento de las células tumorales en HR en relación con hipoxia permitirá realizar planeaciones dosimétricas más dinámicas, que reevalúen las condiciones de oxigenación tumoral constantemente.

Avaluació de la toxicitat de metalls pesants i arsènic en diferents models biològics

En aquest estudi, la toxicitat de diversos metalls pesants i l'arsènic va ser analitzada utilitzant diferents models biològics. En la primera part d'aquest treball, el bioassaig de toxicitat Microtox, el qual està basat en la variació de l'emissió lumínica del bacteri luminiscent Vibrio fischeri, va ser utilitzat per establir les corbes dosi-resposta de diferents elements tòxics com el Zn(II), Pb(II), Cu(II), Hg(II), Ag(I), Co(II), Cd(II), Cr(VI), As(V) i As(III) en solucions aquoses. Els experiments es varen portar a terme a pH 6.0 i 7.0 per tal de mostrar que el pH pot influir en la toxicitat final mesurada d'alguns metalls degut als canvis relacionats amb la seva especiació química. Es varen trobar diferents tipus de corbes dosi-resposta depenent del metall analitzat i el pH del medi. En el cas de l'arsènic, l'efecte del pH en la toxicitat de l'arsenat i l'arsenit es va investigar utilitzant l'assaig Microtox en un rang de pHs comprès entre pH 5.0 i 9.0. Els valors d'EC50 determinats per l'As(V) disminueixen, reflectint un augment de la toxicitat, a mesura que el pH de la solució augmenta mentre que, en el cas de l'As(III), els valors d'EC50 quasi bé no varien entre pH 6.0 i 8.0 i només disminueixen a pH 9.0. HAsO42- i H2AsO3- es varen definir com les espècies més tòxiques. Així mateix, una anàlisi estadística va revelar un efecte antagònic entre les espècies químiques d'arsenat que es troben conjuntament a pH 6.0 i 7.0. D'altra banda, els resultats de dos mètodes estadístics per predir la toxicitat i les possibles interaccions entre el Co(II), Cd(II), Cu(II), Zn(II) i Pb(II) en mescles binàries equitòxiques es varen comparar amb la toxicitat observada sobre el bacteri Vibrio fischeri. L'efecte combinat d'aquests metalls va resultar ser antagònic per les mescles de Co(II)-Cd(II), Cd(II)-Zn(II), Cd(II)-Pb(II) i Cu(II)-Pb(II), sinèrgic per Co(II)-Cu(II) i Zn(II)-Pb(II) i additiu en els altres casos, revelant un patró complex de possibles interaccions. L'efecte sinèrgic de la combinació Co(II)-Cu(II) i la forta disminució de la toxicitat del Pb(II) quan es troba en presència de Cd(II) hauria de merèixer més atenció quan s'estableixen les normatives de seguretat ambiental. La sensibilitat de l'assaig Microtox també va ser determinada. Els valors d'EC20, els quals representen la toxicitat llindar mesurable, varen ser determinats per cada element individualment i es va veure que augmenten de la següent manera: Pb(II) < Ag(I) < Hg(II)  Cu(II) < Zn(II) < As(V) < Cd(II)  Co(II) < As(III) < Cr(VI). Aquests valors es varen comparar amb les concentracions permeses en aigues residuals industrials establertes per la normativa oficial de Catalunya (Espanya). L'assaig Microtox va resultar ser suficientment sensible per detectar els elements assajats respecte a les normes oficials referents al control de la contaminació, excepte en el cas del cadmi, mercuri, arsenat, arsenit i cromat. En la segona part d'aquest treball, com a resultats complementaris dels resultats previs obtinguts utilitzant l'assaig de toxicitat aguda Microtox, els efectes crònics del Cd(II), Cr(VI) i As(V) es varen analitzar sobre la taxa de creixement i la viabilitat en el mateix model biològic. Sorprenentment, aquests productes químics nocius varen resultar ser poc tòxics per aquest bacteri quan es mesura el seu efecte després de temps d'exposició llargs. Tot i això, en el cas del Cr(VI), l'assaig d'inhibició de la viabilitat va resultar ser més sensible que l'assaig de toxicitat aguda Microtox. Així mateix, també va ser possible observar un clar fenomen d'hormesis, especialment en el cas del Cd(II), quan s'utilitza l'assaig d'inhibició de la viabilitat. A més a més, diversos experiments es varen portar a terme per intentar explicar la manca de toxicitat de Cr(VI) mostrada pel bacteri Vibrio fischeri. La resistència mostrada per aquest bacteri podria ser atribuïda a la capacitat d'aquest bacteri de convertir el Cr(VI) a la forma menys tòxica de Cr(III). Es va trobar que aquesta capacitat de reducció depèn de la composició del medi de cultiu, de la concentració inicial de Cr(VI), del temps d'incubació i de la presència d'una font de carboni. En la tercera part d'aquest treball, la línia cel·lular humana HT29 i cultius primaris de cèl·lules sanguínies de Sparus sarba es varen utilitzar in vitro per detectar la toxicitat llindar de metalls mesurant la sobreexpressió de proteines d'estrès. Extractes de fangs precedents de diverses plantes de tractament d'aigues residuals i diferents metalls, individualment o en combinació, es varen analitzar sobre cultius cel·lulars humans per avaluar el seu efecte sobre la taxa de creixement i la capacitat d'induir la síntesi de les proteïnes Hsp72 relacionades amb l'estrès cel·lular. No es varen trobar efectes adversos significatius quan els components s'analitzen individualment. Nogensmenys, quan es troben conjuntament, es produeix un afecte advers sobre tan la taxa de creixement com en l'expressió de proteins d'estrès. D'altra banda, cèl·lules sanguínies procedents de Sparus sarba es varen exposar in vitro a diferents concentracions de cadmi, plom i crom. La proteïna d'estrès HSP70 es va sobreexpressar significativament després de l'exposició a concentracions tan febles com 0.1 M. Sota les nostres condicions de treball, no es va evidenciar una sobreexpressió de metal·lotioneïnes. Nogensmenys, les cèl·lules sanguínies de peix varen resultar ser un model biològic interessant per a ser utilitzat en anàlisis de toxicitat. Ambdós models biològics varen resultar ser molt adequats per a detectar acuradament la toxicitat produïda per metalls. En general, l'avaluació de la toxicitat basada en l'anàlisi de la sobreexpressió de proteïnes d'estrès és més sensible que l'avaluació de la toxicitat realitzada a nivell d'organisme. A partir dels resultats obtinguts, podem concloure que una bateria de bioassaigs és realment necessària per avaluar acuradament la toxicitat de metalls ja que existeixen grans variacions entre els valors de toxicitat obtinguts emprant diferents organismes i molts factors ambientals poden influir i modificar els resultats obtinguts.

Isolation of lactoperoxidase using different cation exchange resins by batch and column procedures

Lactoperoxidase (LP) was isolated from whey protein by cation-exchange using Carboxymethyl resin (CM-25C) and Sulphopropyl Toyopearl resin (SP-650C). Both batch and column procedures were employed and the adsorption capacities and extraction efficiencies were compared. The resin bed volume to whey volume ratios were 0.96:1.0 for CM-25C and ≤ 0.64:1.0 for SP-650 indicating higher adsorption capacity of SP-650 compared to CM-25C. The effluent LP activity depended on both the enzyme activity in the whey and the amount of whey loaded on the column within the saturation limits of the resin. The percentage recovery was high below the saturation point and fell off rapidly with over-saturation. While effective recovery was achieved with column extraction procedures, the recovery was poor in batch procedures. The whey-resin contact time had little impact on the enzyme adsorption. SDS PAGE and HPLC analyses were also carried out, the purity was examined and the proteins characterised in terms of molecular weights. Reversed phase HPLC provided clear distinction of the LP and lactoferrin (LF) peaks. The enzyme purity was higher in column effluents compared to batch effluents, judged on the basis of the clarity of the gel bands and the resolved peaks in HPLC chromatograms.

Germin is a manganese containing homohexamer with oxalate oxidase and superoxide dismutase activities

Germin is a hydrogen peroxide generating oxalate oxidase with extreme thermal stability; it is involved in the defense against biotic and abiotic stress in plants. The structure, determined at 1.6 A resolution, comprises beta-jellyroll monomers locked into a homohexamer (a trimer of dimers), with extensive surface burial accounting for its remarkable stability. The germin dimer is structurally equivalent to the monomer of the 7S seed storage proteins (vicilins), indicating evolution from a common ancestral protein. A single manganese ion is bound per germin monomer by ligands similar to those of manganese superoxide dismutase (MnSOD). Germin is also shown to have SOD activity and we propose that the defense against extracellular superoxide radicals is an important additional role for germin and related proteins.

Microbial relatives of seed storage proteins: conservation of motifs in a functionally diverse superfamily of enzymes

Plant storage proteins comprise a major part of the human diet. Sequence analysis has revealed that these proteins probably share a common ancestor with a fungal oxalate decarboxylase and/or related bacterial genes. Additionally, all these proteins share a central core sequence with several other functionally diverse enzymes and binding proteins, many of which are associated with synthesis of the extracellular matrix during sporulation/encystment. A possible prokaryotic relative of this sequence is a bacterial protein (SASP) known to bind to DNA and thereby protect spores from extreme environmental conditions. This ability to maintain cell viability during periods of dehydration in spores and seeds may relate to absolute conservation of residues involved in structure determination.

In planta proteomics and proteogenomics of the biotrophic barley fungal pathogen blumeria graminis f. sp hordei

To further our understanding of powdery mildew biology during infection, we undertook a systematic shotgun proteomics analysis of the obligate biotroph Blumeria graminis f. sp. hordei at different stages of development in the host. Moreover we used a proteogenomics approach to feed information into the annotation of the newly sequenced genome. We analyzed and compared the proteomes from three stages of development representing different functions during the plant-dependent vegetative life cycle of this fungus. We identified 441 proteins in ungerminated spores, 775 proteins in epiphytic sporulating hyphae, and 47 proteins from haustoria inside barley leaf epidermal cells and used the data to aid annotation of the B. graminis f. sp. hordei genome. We also compared the differences in the protein complement of these key stages. Although confirming some of the previously reported findings and models derived from the analysis of transcriptome dynamics, our results also suggest that the intracellular haustoria are subject to stress possibly as a result of the plant defense strategy, including the production of reactive oxygen species. In addition, a number of small haustorial proteins with a predicted N-terminal signal peptide for secretion were identified in infected tissues: these represent candidate effector proteins that may play a role in controlling host metabolism and immunity. Molecular & Cellular Proteomics 8: 2368-2381, 2009.

Proteomic analysis of resting and thrombin-stimulated platelets reveals the translocation and functional relevance of HIP-55 in platelets

The platelet surface is a dynamic interface that changes rapidly in response to stimuli to coordinate the formation of thrombi at sites of vascular injury. Tight control is essential as loss of organisation may result in the inappropriate formation of thrombi (thrombosis) or excessive bleeding. In this paper we describe the comparative analysis of resting and thrombin-stimulated platelet membrane proteomes and associated proteins to identify proteins important to platelet function. Surface proteins were labelled using a biotin tag and isolated by NeurtrAvidin affinity chromatography. Liquid phase IEF and SDS-PAGE were used to separate proteins, and bands of increased intensity in the stimulated platelet fractions were digested and identified by FT-ICR mass spectrometry. Novel proteins were identified along with proteins known to be translocated to the platelet surface. Furthermore, many platelet proteins revealed changes in location associated with function, including G6B and Hip-55. HIP-55 is an SH3-binding protein important in T-cell receptor signalling. Further analysis of HIP-55 revealed that this adaptor protein becomes increasingly associated with both Syk and integrin beta 3 upon platelet activation. Analysis of HIP-55 deficient platelets revealed reduced fibrinogen binding upon thrombin stimulation, suggesting HIP-55 to be an important regulator of platelet function.

Assembly and maintenance of the sarcomere night and day

The assembly of sarcomeric proteins into the highly organized structure of the sarcomere is an ordered and complex process involving an array of structural and associated proteins. The sarcomere has shown itself to be considerably more complex than ever envisaged and may be considered one of the most complex macromolecular assemblies in biology. Studies over the last decade have helped to put a new face on the sarcomere, and, as such, the sarcomere is being redefined as a dynamic network of proteins capable of generating force and signalling with other cellular compartments and metabolic enzymes capable of controlling many facets of striated myocyte biology.

Flavonoids inhibit the platelet TxA(2) signalling pathway and antagonize TxA(2) receptors (TP) in platelets and smooth muscle cells

What is already known about this subject center dot Flavonoids are largely recognized as potential inhibitors of platelet function, through nonspecific mechanisms such as antioxidant activity and/or inhibition of several enzymes and signalling proteins. center dot In addition, we, and few others, have shown that certain antiaggregant flavonoids may behave as specific TXA2 receptor (TP) ligands in platelets. center dot Whether flavonoids interact with TP isoforms in other cell types is not known, and direct evidence that flavonoid-TP interaction inhibits signalling downstream TP has not been shown. What this study adds center dot This study first demonstrates that certain flavonoids behave as ligands for both TP isoforms, not only in platelets, but also in human myometrium and in TP-transfected HEK 293T cells. center dot Differences in the effect of certain flavonoids in platelet signalling, induced by either U46619 or thrombin, suggest that abrogation of downstream TP signalling is related to their specific blockage of the TP, rather than to a nonspecific effect on tyrosine kinases or other signalling proteins. Flavonoids may affect platelet function by several mechanisms, including antagonism of TxA(2) receptors (TP). These TP are present in many tissues and modulate different signalling cascades. We explored whether flavonoids affect platelet TP signalling, and if they bind to TP expressed in other cell types. Platelets were treated with flavonoids, or other selected inhibitors, and then stimulated with U46619. Similar assays were performed in aspirinized platelets activated with thrombin. Effects on calcium release were analysed by fluorometry and changes in whole protein tyrosine phosphorylation and activation of ERK 1/2 by Western blot analysis. The binding of flavonoids to TP in platelets, human myometrium and TP alpha- and TP beta-transfected HEK 293T cells was explored using binding assays and the TP antagonist H-3-SQ29548. Apigenin, genistein, luteolin and quercetin impaired U46619-induced calcium mobilization in a concentration-dependent manner (IC50 10-30 mu M). These flavonoids caused a significant impairment of U46619-induced platelet tyrosine phosphorylation and of ERK 1/2 activation. By contrast, in aspirin-treated platelets all these flavonoids, except quercetin, displayed minor effects on thrombin-induced calcium mobilization, ERK 1/2 and total tyrosine phosphorylation. Finally, apigenin, genistein and luteolin inhibited by > 50% H-3-SQ29548 binding to different cell types. These data further suggest that flavonoids may inhibit platelet function by binding to TP and by subsequent abrogation of downstream signalling. Binding of these compounds to TP occurs in human myometrium and in TP-transfected HEK 293T cells and suggests that antagonism of TP might mediate the effects of flavonoids in different tissues.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine (DCVC) resulted in a >1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

Ovarian follicle development in the laying hen is accompanied by divergent changes in inhibin A, inhibin B, activin A and follistatin production in granulosa and theca layers

To study the potential involvement of inhibin A (inhA), inhibin B (inhB), activin A (actA) and follistatin (FS) in the recruitment of follicles into the preovulatory hierarchy, growing follicles (ranging from 1 mm to the largest designated F1) and the three most recent postovulatory follicles (POFs) were recovered from laying hens (n=11). With the exception of <4 mm follicles and POFs, follicle walls were dissected into separate granulosa (G) and theca (T) layers before extraction. Contents of inhA, inhB, actA and FS in tissue extracts were assayed using specific two-site ELISAs and results are expressed per mg DNA. InhB content of both G and T followed a similar developmental pattern, although the content was >4-fold higher in G than in T at all stages. InhB content was very low in follicles <4 nun but increased - 50-fold (P<0.0001) to peak in 7-9 mm follicles, before falling steadily as follicles entered and moved up the follicular hierarchy (40-fold; 8 mm vs F2). In stark contrast, inhA remained very low in prehierarchical follicles (&LE; 9 mm) but then increased progressively as follicles moved up the preovulatory hierarchy to peak in F1 (&SIM; 100-fold increase; P<0.0001); In F1 >97% of inhA was confined to the G layer whereas in 5-9 mm follicles inhA was only. detected in the T layer. Both inhA and inhB contents of POFs were significantly reduced compared with F1. Follicular actA was mainly confined to the T layer although detectable levels were present in G from 9 nun; actA was low between 1 and 9 mm but increased sharply as follicles entered the preovulatory hierarchy (&SIM;6-fold higher in F4; P<0.0001); levels then fell &SIM;2-fold as the follicle progressed to F1. Like actA, FS predominated in the T although significant amounts were also present in the G of prehierarchical follicles (4-9 mm), in contrast to actA, which was absent from the G. The FS content of T rose &SIM;3-fold from 6 mm to a plateau which was sustained until F1. In contrast, the FS content of G was greatest in prehierarchical follicles and fell &SIM;4-fold in F4-F1 follicles. ActA and FS contents of POFs were reduced compared with F1. In vitro studies on follicle wall explants confirmed the striking divergence in the secretion of inhA and inhB during follicle development. These findings of marked stage-dependent differences in the expression of inhA, inhB, actA and FS proteins imply a significant functional role for these peptides in the recruitment and ordered progression of follicles within the avian ovary.

In planta proteomics and proteogenomics of the biotrophic barley fungal pathogen blumeria graminis f. sp hordei

To further our understanding of powdery mildew biology during infection, we undertook a systematic shotgun proteomics analysis of the obligate biotroph Blumeria graminis f. sp. hordei at different stages of development in the host. Moreover we used a proteogenomics approach to feed information into the annotation of the newly sequenced genome. We analyzed and compared the proteomes from three stages of development representing different functions during the plant-dependent vegetative life cycle of this fungus. We identified 441 proteins in ungerminated spores, 775 proteins in epiphytic sporulating hyphae, and 47 proteins from haustoria inside barley leaf epidermal cells and used the data to aid annotation of the B. graminis f. sp. hordei genome. We also compared the differences in the protein complement of these key stages. Although confirming some of the previously reported findings and models derived from the analysis of transcriptome dynamics, our results also suggest that the intracellular haustoria are subject to stress possibly as a result of the plant defense strategy, including the production of reactive oxygen species. In addition, a number of small haustorial proteins with a predicted N-terminal signal peptide for secretion were identified in infected tissues: these represent candidate effector proteins that may play a role in controlling host metabolism and immunity. Molecular & Cellular Proteomics 8: 2368-2381, 2009.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine ( DCVC) resulted in a > 1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

Dietary, physiological, genetic and pathological influences on postprandial lipid metabolism

Most of diurnal time is spent in a postprandial state due to successive meal intakes during the day. As long as the meals contain enough fat, a transient increase in triacylglycerolaemia and a change in lipoprotein pattern occurs. The extent and kinetics of such postprandial changes are highly variable and are modulated by numerous factors. This review focuses on factors affecting postprandial lipoprotein metabolism and genes, their variability and their relationship with intermediate phenotypes and risk of CHD. Postprandial lipoprotein metabolism is modulated by background dietary pattern as well as meal composition (fat amount and type, carbohydrate, protein, fibre, alcohol) and several lifestyle conditions (physical activity, tobacco use), physiological factors (age, gender, menopausal status) and pathological conditions (obesity, insulin resistance, diabetes mellitus). The roles of many genes have been explored in order to establish the possible implications of their variability in lipid metabolism and CHD risk. The postprandial lipid response has been shown to be modified by polymorphisms within the genes for apo A-I, A-IV, AN, E, B, C-I and C-III, lipoprotein lipase, hepatic lipase, fatty acid binding and transport proteins, microsomal trigyceride transfer protein and scavenger receptor class B type I. Overall, the variability in postprandial response is important and complex, and the interactions between nutrients or dietary or meal compositions and gene variants need further investigation. The extent of present knowledge and needs for future studies are discussed in light of ongoing developments in nutrigenetics.

Recovery of lactoferrin and lactoperoxidase from sweet whey using colloidal gas aphrons (CGAs) generated from an anionic surfactant, AOT

The recovery of lactoferrin and lactoperoxidase from sweet whey was studied using colloidal gas aphrons (CGAs), which are surfactant-stabilized microbubbles (10-100 mum). CGAs are generated by intense stirring (8000 rpm for 10 min) of the anionic surfactant AOT (sodium bis-2-ethylhexyl sulfosuccinate). A volume of CGAs (10-30 mL) is mixed with a given volume of whey (1 - 10 mL), and the mixture is allowed to separate into two phases: the aphron (top) phase and the liquid (bottom) phase. Each of the phases is analyzed by SDS-PAGE and surfactant colorimetric assay. A statistical experimental design has been developed to assess the effect of different process parameters including pH, ionic strength, the concentration of surfactant in the CGAs generating solution, the volume of CGAs and the volume of whey on separation efficiency. As expected pH, ionic strength and the volume of whey (i.e. the amount of total protein in the starting material) are the main factors influencing the partitioning of the Lf(.)Lp fraction into the aphron phase. Moreover, it has been demonstrated that best separation performance was achieved at pH = 4 and ionic strength = 0.1 mol/L i.e., with conditions favoring electrostatic interactions between target proteins and CGAs (recovery was 90% and the concentration of lactoferrin and lactoperoxidase in the aphron phase was 25 times higher than that in the liquid phase), whereas conditions favoring hydrophobic interactions (pH close to pI and high ionic strength) led to lower performance. However, under these conditions, as confirmed by zeta potential measurements, the adsorption of both target proteins and contaminant proteins is favored. Thus, low selectivity is achieved at all of the studied conditions. These results confirm the initial hypothesis that CGAs act as ion exchangers and that the selectivity of the process can be manipulated by changing main operating parameters such as type of surfactant, pH and ionic strength.