High-efficiency oxygen evolution by photosystem II oxygen-evolving complex containing 3Mn per reaction center.

Ca-depleted photosystem II membranes obtained by treatment with acidic buffer do not contain Ca2+ in the Mn4CaO5 cluster but contain all extrinsic proteins protecting this cluster (PSII(-Ca/low pH)). However, unlike native photosystem II, Mn cluster in PSII(-Ca/low pH) samples is available for small-sized reductants. Using this property, we investigated the substitution possibility of Mn cation(s) with Fe cation(s) to obtain a chimeric cluster in PSII(-Ca/low pH) samples containing extrinsic proteins. We found that Fe(II) cation replaces Mn cation at pH 6.5, however, PSII(-Ca/low pH) membranes with the 3Mn1Fe chimeric cluster in the oxygen-evolving complex evolve O2 with high intensity in the presence of exogenous Ca2+. The O2 evolution rate is about 80% of the same rate in PSII(-Ca/low pH) membranes.

Binding and functions of the two chloride ions in the oxygen-evolving center of photosystem II.

Light-driven water oxidation in photosynthesis occurs at the oxygen-evolving center (OEC) of photosystem II (PSII). Chloride ions (Cl-) are essential for oxygen evolution by PSII, and two Cl- ions have been found to specifically bind near the Mn4CaO5 cluster in the OEC. The retention of these Cl- ions within the OEC is critically supported by some of the membrane-extrinsic subunits of PSII. The functions of these two Cl- ions and the mechanisms of their retention both remain to be fully elucidated. However, intensive studies performed recently have advanced our understanding of the functions of these Cl- ions, and PSII structures from various species have been reported, aiding the interpretation of previous findings regarding Cl- retention by extrinsic subunits. In this review, we summarize the findings to date on the roles of the two Cl- ions bound within the OEC. Additionally, together with a short summary of the functions of PSII membrane-extrinsic subunits, we discuss the mechanisms of Cl- retention by these extrinsic subunits.

The PsbJ protein is required for photosystem II activity in centers lacking the PsbO and PsbV lumenal subunits.

Photosystem II (PS II) of oxygenic photosynthesis is found in the thylakoid membranes of plastids and cyanobacteria. The mature PS II complex comprises a central core of four membrane proteins that bind the majority of the redox-active cofactors. In cyanobacteria the central core is surrounded by 13 low-molecular-weight (LMW) subunits which each consist of one or two transmembrane helices. Three additional hydrophilic subunits known as PsbO, PsbU and PsbV are found associated with hydrophilic loops belonging to the core proteins protruding into the thylakoid lumen. During biogenesis the majority of the LMW subunits are known to initially associate with individual pre-assembly complexes consisting of one or more of the core proteins; however, the point at which the PsbJ LMW subunit binds to PS II is not known. The majority of models for PS II biogenesis propose that the three extrinsic proteins and PsbJ bind in the final stages of PS II assembly. We have investigated the impact of creating the double mutants ∆PsbJ:∆PsbO, ∆PsbJ:∆PsbU and ∆PsbJ:∆PsbV to investigate potential cooperation between these subunits in the final stages of biogenesis. Our results indicate that PsbJ can bind to PS II in the absence of any one of the extrinsic proteins. However, unlike their respective single mutants, the ∆PsbJ:∆PsbO and ∆PsbJ:∆PsbV strains were not photoautotrophic and were unable to support oxygen evolution suggesting a functional oxygen-evolving complex could not assemble in these strains. In contrast, the PS II centers formed in the ∆PsbJ:∆PsbU strain were capable of photoautotrophic growth and could support oxygen evolution when whole-chain electron transport was supported by the addition of bicarbonate.

D139N mutation of PsbP enhances the oxygen-evolving activity of photosystem II through stabilized binding of a chloride ion.

Photosystem II (PSII) is a multisubunit membrane protein complex that catalyzes light-driven oxidation of water to molecular oxygen. The chloride ion (Cl-) has long been known as an essential cofactor for oxygen evolution by PSII, and two Cl- ions (Cl-1 and Cl-2) have been found to specifically bind near the Mn4CaO5 cluster within the oxygen-evolving center (OEC). However, despite intensive studies on these Cl- ions, little is known about the function of Cl-2, the Cl- ion that is associated with the backbone nitrogens of D1-Asn338, D1-Phe339, and CP43-Glu354. In green plant PSII, the membrane extrinsic subunits-PsbP and PsbQ-are responsible for Cl- retention within the OEC. The Loop 4 region of PsbP, consisting of highly conserved residues Thr135-Gly142, is inserted close to Cl-2, but its importance has not been examined to date. Here, we investigated the importance of PsbP-Loop 4 using spinach PSII membranes reconstituted with spinach PsbP proteins harboring mutations in this region. Mutations in PsbP-Loop 4 had remarkable effects on the rate of oxygen evolution by PSII. Moreover, we found that a specific mutation, PsbP-D139N, significantly enhances the oxygen-evolving activity in the absence of PsbQ, but not significantly in its presence. The D139N mutation increased the Cl- retention ability of PsbP and induced a unique structural change in the OEC, as indicated by light-induced Fourier transform infrared (FTIR) difference spectroscopy and theoretical calculations. Our findings provide insight into the functional significance of Cl-2 in the water-oxidizing reaction of PSII.

Role of PsbV-Tyr137 in photosystem II studied by site-directed mutagenesis in the thermophilic cyanobacterium Thermosynechococcus vulcanus.

PsbV (cytochrome c550) is one of the three extrinsic proteins of photosystem II (PSII) and functions to maintain the stability and activity of the Mn4CaO5 cluster, the catalytic center for water oxidation. PsbV-Y137 is the C-terminal residue of PsbV and is located at the exit of a hydrogen-bond network mediated by the D1-Y161-H190 residue pair. In order to examine the function of PsbV-Y137, four mutants, PsbV-Y137A, PsbV-Y137F, PsbV-Y137G, and PsbV-Y137W, were generated with Thermosynechococcus vulcanus (T. vulcanus). These mutants showed growth rates similar to that of the wild-type strain (WT); however, their oxygen-evolving activities were different. At pH 6.5, the oxygen evolution rates of Y137F and Y137W were almost identical to that of WT, whereas the oxygen evolution rates of the Y137A, Y137G mutants were 64% and 61% of WT, respectively. However, the oxygen evolution in the latter two mutants decreased less at higher pHs, suggesting that higher pHs facilitated oxygen evolution probably by facilitating proton egress in these two mutants. Furthermore, thylakoid membranes isolated from the PsbV-Y137A, PsbV-Y137G mutants exhibited much lower levels of oxygen-evolving activity than that of WT, which was found to be caused by the release of PsbV. In addition, PSII complexes purified from the PsbV-Y137A and PsbV-Y137G mutants lost all of the three extrinsic proteins but instead bind Psb27, an assembly cofactor of PSII. These results demonstrate that the PsbV-Tyr137 residue is required for the stable binding of PsbV to PSII, and the hydrogen-bond network mediated by D1-Y161-H190 is likely to function in proton egress during water oxidation.

Photosystem II Extrinsic Proteins and Their Putative Role in Abiotic Stress Tolerance in Higher Plants.

Abiotic stress remains one of the major challenges in managing and preventing crop loss. Photosystem II (PSII), being the most susceptible component of the photosynthetic machinery, has been studied in great detail over many years. However, much of the emphasis has been placed on intrinsic proteins, particularly with respect to their involvement in the repair of PSII-associated damage. PSII extrinsic proteins include PsbO, PsbP, PsbQ, and PsbR in higher plants, and these are required for oxygen evolution under physiological conditions. Changes in extrinsic protein expression have been reported to either drastically change PSII efficiency or change the PSII repair system. This review discusses the functional role of these proteins in plants and indicates potential areas of further study concerning these proteins.

Electrostatic interaction of positive charges on the surface of Psb31 with photosystem II in the diatom Chaetoceros gracilis.

Psb31, a novel extrinsic protein found in diatom photosystem II (PSII), directly binds to PSII core subunits, independent of the other extrinsic proteins, and functions to maintain optimum oxygen evolution. However, how Psb31 electrostatically interacts with PSII intrinsic proteins remains to be clarified. In this study, we examined electrostatic interaction of Psb31 with PSII complexes isolated from the diatom Chaetoceros gracilis. Positive or negative charges of isolated Psb31 proteins were modified with N-succinimidyl propionate (NSP) or glycine methyl ester (GME), respectively, resulting in formation of uncharged groups. NSP-modified Psb31 did not bind to PSII with a concomitant increase in NSP concentration, whereas GME-modified Psb31 clearly bound to PSII with retention of oxygen-evolving activity, indicating that positive charges of Lys residues and the N-terminus on the surface of Psb31 are involved in electrostatic interactions with PSII intrinsic proteins. Mass spectrometry analysis of NSP-modified Psb31 and sequence comparisons of Psb31 from C. gracilis with other chromophyte algae led to identification of three Lys residues as possible binding sites to PSII. Based on these findings, together with our previous cross-linking study in diatom PSII and a red algal PSII structure, we discuss binding properties of Psb31 with PSII core proteins.

Structurally conserved channels in cyanobacterial and plant photosystem II.

In the cyanobacterial photosystem II (PSII), the O4-water chain in the D1 and CP43 proteins, a chain of water molecules that are directly H-bonded to O4 of the Mn4Ca cluster, is linked with a channel that connects the protein bulk surface along with a membrane-extrinsic protein subunit, PsbU (O4-PsbU channel). The cyanobacterial PSII structure also shows that the O1 site of the Mn4Ca cluster has a chain of H-bonded water molecules, which is linked with the channel that proceeds toward the bulk surface via PsbU and PsbV (O1-PsbU/V channel). Membrane-extrinsic protein subunits PsbU and PsbV in cyanobacterial PSII are replaced with PsbP and PsbQ in plant PSII. However, these four proteins have no structural similarity. It remains unknown whether the corresponding channels also exist in plant PSII, because water molecules are not identified in the plant PSII cryo-electron microscopy (cryo-EM) structure. Using the cyanobacterial and plant PSII structures, we analyzed the channels that proceed from the Mn4Ca cluster. The cyanobacterial O4-PsbU and O1-PsbU/V channels were structurally conserved as the channel that proceeds along PsbP toward the protein bulk surface in the plant PSII (O4-PsbP and O1-PsbP channels, respectively). Calculated protonation states indicated that in contrast to the original geometry of the plant cryo-EM structure, protonated PsbP-Lys166 may form a salt-bridge with ionized D1-Glu329 and protonated PsbP-Lys173 may form a salt-bridge with ionized PsbQ-Asp28 near the O1-PsbP channel. The existence of these channels might explain the molecular mechanism of how PsbP can interact with the Mn4Ca cluster.

Environmental pH and the Requirement for the Extrinsic Proteins of Photosystem II in the Function of Cyanobacterial Photosynthesis.

In one of the final stages of cyanobacterial Photosystem II (PS II) assembly, binding of up to four extrinsic proteins to PS II stabilizes the oxygen-evolving complex (OEC). Growth of cyanobacterial mutants deficient in certain combinations of these thylakoid-lumen-associated polypeptides is sensitive to changes in environmental pH, despite the physical separation of the membrane-embedded PS II complex from the external environment. In this perspective we discuss the effect of environmental pH on OEC function and photoautotrophic growth in cyanobacteria with reference to pH-sensitive PS II mutants lacking extrinsic proteins. We consider the possibilities that, compared to pH 10.0, pH 7.5 increases susceptibility to PS II-generated reactive oxygen species (ROS) causing photoinhibition and reducing PS II assembly in some mutants, and that perturbations to channels in the lumenal regions of PS II might alter the accessibility of water to the active site as well as egress of oxygen and protons to the thylakoid lumen. Reduced levels of PS II in these mutants, and reduced OEC activity arising from the disruption of substrate/product channels, could reduce the trans-thylakoid pH gradient (ΔpH), leading to the impairment of photosynthesis. Growth of some PS II mutants at pH 7.5 can be rescued by elevating CO2 levels, suggesting that the pH-sensitive phenotype might primarily be an indirect result of back-pressure in the electron transport chain that results in heightened production of ROS by the impaired photosystem.

Manganese Deficiency in Plants: The Impact on Photosystem II.

Manganese (Mn) is an essential plant micronutrient with an indispensable function as a catalyst in the oxygen-evolving complex (OEC) of photosystem II (PSII). Even so, Mn deficiency frequently occurs without visual leaf symptoms, thereby masking the distribution and dimension of the problem restricting crop productivity in many places of the world. Hence, timely alleviation of latent Mn deficiency is a challenge in promoting plant growth and quality. We describe here the key mechanisms of Mn deficiency in plants by focusing on the impact of Mn on PSII stability and functionality. We also address the mechanisms underlying the differential tolerance towards Mn deficiency observed among plant genotypes, which enable Mn-efficient plants to grow on marginal land with poor Mn availability.

Structural Coupling of Extrinsic Proteins with the Oxygen-Evolving Center in Photosystem II.

Photosystem II (PSII), which catalyzes photosynthetic water oxidation, is composed of more than 20 subunits, including membrane-intrinsic and -extrinsic proteins. The PSII extrinsic proteins shield the catalytic Mn4CaO5 cluster from the outside bulk solution and enhance binding of inorganic cofactors, such as Ca(2+) and Cl(-), in the oxygen-evolving center (OEC) of PSII. Among PSII extrinsic proteins, PsbO is commonly found in all oxygenic organisms, while PsbP and PsbQ are specific to higher plants and green algae, and PsbU, PsbV, CyanoQ, and CyanoP exist in cyanobacteria. In addition, red algae and diatoms have unique PSII extrinsic proteins, such as PsbQ' and Psb31, suggesting functional divergence during evolution. Recent studies with reconstitution experiments combined with Fourier transform infrared spectroscopy have revealed how the individual PSII extrinsic proteins affect the structure and function of the OEC in different organisms. In this review, we summarize our recent results and discuss changes that have occurred in the structural coupling of extrinsic proteins with the OEC during evolutionary history.

A laser ablation ICP-MS based method for multiplexed immunoblot analysis: applications to manganese-dependent protein dynamics of photosystem II in barley (Hordeum vulgare L.).

Manganese (Mn) constitutes an essential co-factor in the oxygen-evolving complex of photosystem II (PSII). Consequently, Mn deficiency reduces photosynthetic efficiency and leads to changes in PSII composition. In order to study these changes, multiplexed protein assays are advantageous. Here, we developed a multiplexed antibody-based assay and analysed selected PSII subunits in barley (Hordeum vulgare L.). A selection of antibodies were labelled with specific lanthanides and immunoreacted with thylakoids exposed to Mn deficiency after western blotting. Subsequently, western blot membranes were analysed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), which allowed selective and relative quantitative analysis via the different lanthanides. The method was evaluated against established liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methods, based on data-dependent acquisition (DDA) and selected reaction monitoring (SRM). Manganese deficiency resulted in a general decrease in PSII protein abundances, an effect that was shown to be reversible upon Mn re-supplementation. Specifically, the extrinsic proteins PsbP and PsbQ showed Mn-dependent changes in abundances. Similar trends in the response to Mn deficiency at the protein level were observed when comparing DDA, SRM and LA-ICP-MS results. A biologically important exception to this trend was the loss of PsbO in the SRM analysis, which highlights the necessity of validating protein changes by more than one technique. The developed method enables a higher number of proteins to be multiplexed in comparison to existing immunoassays. Furthermore, multiplexed protein analysis by LA-ICP-MS provides an analytical platform with high throughput appropriate for screening large collections of plants.

Localization and functional characterization of the extrinsic subunits of photosystem II: an update.

Photosystem II (PSII), which catalyzes photosynthetic water oxidation, is composed of more than 20 subunits, including membrane-intrinsic and -extrinsic proteins. The extrinsic proteins of PSII shield the catalytic Mn4CaO5 cluster from exogenous reductants and serve to optimize oxygen evolution at physiological ionic conditions. These proteins include PsbO, found in all oxygenic organisms, PsbP and PsbQ, specific to higher plants and green algae, and PsbU, PsbV, CyanoQ, and CyanoP in cyanobacteria. Furthermore, red algal PSII has PsbQ' in addition to PsbO, PsbV, and PsbU, and diatoms have Psb31 in supplement to red algal-type extrinsic proteins, exemplifying the functional divergence of these proteins during evolution. This review provides an updated summary of recent findings on PSII extrinsic proteins and discusses their binding, function, and evolution within various photosynthetic organisms.

The PsbP and PsbQ family proteins in the photosynthetic machinery of chloroplasts.

The PsbP and PsbQ proteins are extrinsic subunits of the photosystem II in eukaryotic photosynthetic organisms including higher plants, green algae and euglena. It has been suggested that PsbP and PsbQ have evolved from their cyanobacterial homologs, while considerable genetic and functional modifications have occurred to generate the eukaryote-type proteins. In addition, number of PsbP and PsbQ homologs exist in the thylakoid lumen of chloroplasts. These homologs are nuclear-encoded and likely diverged by gene duplication, and recent studies have elucidated their various functions in the photosynthetic machinery. In this short review, recent findings and new idea about these components will be discussed.

Environmental pH affects photoautotrophic growth of Synechocystis sp. PCC 6803 strains carrying mutations in the lumenal proteins of PSII.

Synechocystis sp. strain PCC 6803 grows photoautotrophically across a broad pH range, but wild-type cultures reach a higher density at elevated pH; however, photoheterotrophic growth is similar at high and neutral pH. A number of PSII mutants each lacking at least one lumenal extrinsic protein, and carrying a second PSII lumenal mutation, are able to grow photoautotrophically in BG-11 medium at pH 10.0, but not pH 7.5. We investigated the basis of this pH effect and observed no pH-specific change in variable fluorescence yield from PSII centers of the wild type or the pH-dependent ΔPsbO:ΔPsbU and ΔPsbV:ΔCyanoQ strains; however, 77 K fluorescence emission spectra indicated increased coupling of the phycobilisome (PBS) antenna at pH 10.0 in all mutants. DNA microarray data showed a cell-wide response to transfer from pH 10.0 to pH 7.5, including decreased mRNA levels of a number of oxidative stress-responsive transcripts. We hypothesize that this transcriptional response led to increased tolerance against reactive oxygen species and in particular singlet oxygen. This response enabled photoautotrophic growth of the PSII mutants at pH 10.0. This hypothesis was supported by increased resistance of all strains to rose bengal at pH 10.0 compared with pH 7.5.

Salazones y chacinados embutidos secos: detección por electroforesis de especies cárnicas y de proteínas extrínsecas agregadas / Dry-cured meat products: electrophoresis detection of meat species and extrinsic proteins added

En la elaboración de salazones y chacinados embutidos secos se pueden utilizar diferentes especies cárnicas y en algunos de ellos también se pueden agregar proteínas extrínsecas. En elpresente trabajo se plantearon los siguientes objetivos: analizar salazones y chacinados embutidos secos elaborados con especies cárnicas de diferente origen (vacuna, porcina, de ciervo, de jabalí y de cordero) para establecer la utilidad de SDS-PAGE en la identificación de las mismas; comparar esta metodología conun método inmunoquímico (ELISA para especie porcina y ELISA para especie vacuna) en muestras que declaraban carne porcina y/o vacuna; detectar la posible presencia de proteínas extrínsecasdeclaradas o no en los respectivos rótulos de estos productos. Se analizaron 5 salazones y 7 chacinados embutidos secos. En todas las muestras se detectaron por electroforesis la o lasespecies cárnicas declaradas, sólo en una muestra que declaraba carnes vacuna y porcina se detectó sólo carne vacuna. Lametodología inmunoquímica confirmó la detección de las carnes vacuna y/o porcina en las muestras que las declaraban. Con respecto a las proteínas extrínsecas se detectaron por electroforesis proteínas de soja en tres muestras, dos de ellas no las declaraban.Se detectaron proteínas lácteas en muy baja concentración en tres muestras que las declaraban y se detectaron proteínas de trigo en dos muestras que no las declaraban. Las tres proteínas detectadas en estas muestras constituyen alergenos alimentarios. Si bien el método ELISA resulta de elección para la detecciónde alergenos alimentarios, ya que tiene una sensibilidad mucho mayor al SDS-PAGE, estas proteínas alergénicas fueron detectadas con facilidad por electroforesis, lo cual indica que estaban agregadas en concentraciones importantes. Resulta entonces imprescindible que los elaboradores de este tipo de productos declaren la totalidad de los ingredientes proteicos utilizados para evitar reacciones alérgicas...

Different meat species can be used in dry-cured meat products, and extrinsic proteins can also be added in some of them. The objectives of this work are to analyze dry-cured meat productselaborated with different meat species (cow, swine, deer, boar and lamb) in order to evaluate the usefulness of the SDS-PAGE method in the identification of the species used; to compare thismethodology with an immunochemical method (ELISA for porcine and bovine species respectively) in samples that declare porcine and/or bovine meat in their label; and to detect the possiblepresence of extrinsic proteins declared or not in these products’ labels. Twelve dry-cured meat products were analyzed. In allsamples, the meat species declared in the labels were found by electrophoresis, except in one case where only bovine meat was detected while the label declared both bovine and porcine meats. The immunochemical method confirmed the detection of bovine and/or porcine meat in the samples that declared them in their labels. As regards extrinsic proteins, soy proteins were detected by electrophoresis in three samples, while two of them did not declare these proteins. Dairy proteins were detected in very low levels in three samples that declared them and wheat proteins were detected in two samples that did notdeclare them. The three proteins detected in these samples are food allergens. Although the method of choice for the detection of allergens in food is ELISA because it is more sensible than SDS-PAGE, these allergenic proteins were easily detected by electrophoresis; this indicates they were added in relevant concentrations. It is then critical that manufacturers declare all proteiningredients used in this kind of products to prevent allergic reactions in consumers.

Salazones y chacinados embutidos secos: detección por electroforesis de especies cárnicas y de proteínas extrínsecas agregadas / Dry-cured meat products: electrophoresis detection of meat species and extrinsic proteins added

En la elaboración de salazones y chacinados embutidos secos se pueden utilizar diferentes especies cárnicas y en algunos de ellos también se pueden agregar proteínas extrínsecas. En elpresente trabajo se plantearon los siguientes objetivos: analizar salazones y chacinados embutidos secos elaborados con especies cárnicas de diferente origen (vacuna, porcina, de ciervo, de jabalí y de cordero) para establecer la utilidad de SDS-PAGE en la identificación de las mismas; comparar esta metodología conun método inmunoquímico (ELISA para especie porcina y ELISA para especie vacuna) en muestras que declaraban carne porcina y/o vacuna; detectar la posible presencia de proteínas extrínsecasdeclaradas o no en los respectivos rótulos de estos productos. Se analizaron 5 salazones y 7 chacinados embutidos secos. En todas las muestras se detectaron por electroforesis la o lasespecies cárnicas declaradas, sólo en una muestra que declaraba carnes vacuna y porcina se detectó sólo carne vacuna. Lametodología inmunoquímica confirmó la detección de las carnes vacuna y/o porcina en las muestras que las declaraban. Con respecto a las proteínas extrínsecas se detectaron por electroforesis proteínas de soja en tres muestras, dos de ellas no las declaraban.Se detectaron proteínas lácteas en muy baja concentración en tres muestras que las declaraban y se detectaron proteínas de trigo en dos muestras que no las declaraban. Las tres proteínas detectadas en estas muestras constituyen alergenos alimentarios. Si bien el método ELISA resulta de elección para la detecciónde alergenos alimentarios, ya que tiene una sensibilidad mucho mayor al SDS-PAGE, estas proteínas alergénicas fueron detectadas con facilidad por electroforesis, lo cual indica que estaban agregadas en concentraciones importantes. Resulta entonces imprescindible que los elaboradores de este tipo de productos declaren la totalidad de los ingredientes proteicos utilizados para evitar reacciones alérgicas...(AU)

Different meat species can be used in dry-cured meat products, and extrinsic proteins can also be added in some of them. The objectives of this work are to analyze dry-cured meat productselaborated with different meat species (cow, swine, deer, boar and lamb) in order to evaluate the usefulness of the SDS-PAGE method in the identification of the species used; to compare thismethodology with an immunochemical method (ELISA for porcine and bovine species respectively) in samples that declare porcine and/or bovine meat in their label; and to detect the possiblepresence of extrinsic proteins declared or not in these products labels. Twelve dry-cured meat products were analyzed. In allsamples, the meat species declared in the labels were found by electrophoresis, except in one case where only bovine meat was detected while the label declared both bovine and porcine meats. The immunochemical method confirmed the detection of bovine and/or porcine meat in the samples that declared them in their labels. As regards extrinsic proteins, soy proteins were detected by electrophoresis in three samples, while two of them did not declare these proteins. Dairy proteins were detected in very low levels in three samples that declared them and wheat proteins were detected in two samples that did notdeclare them. The three proteins detected in these samples are food allergens. Although the method of choice for the detection of allergens in food is ELISA because it is more sensible than SDS-PAGE, these allergenic proteins were easily detected by electrophoresis; this indicates they were added in relevant concentrations. It is then critical that manufacturers declare all proteiningredients used in this kind of products to prevent allergic reactions in consumers.(AU)