Development of a rapid, high-sensitivity, low-cost fluorescence method for protein surface hydrophobicity determination using a Nanodrop fluorospectrometer

Cruz-Torres, Luis F. De la; Rodríguez-Celestino, Verónica; Centeno-Leija, Sara; Serrano‐Posada, Hugo; Ceballos‐Magaña, Silvia G.; Aguilar-Padilla, Jorge; Mancilla-Margalli, N. Alejandra; Osuna-Castro, Juan Alberto

doi:10.1016/j.foodchem.2022.133681

Cited by 8 publications

(5 citation statements)

References 35 publications

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“…The surface hydrophobicity is also affected by the higher structure of proteins. For example, when proteins are subjected to high-temperature, high-pressure, shear force, and other factors, the higher structure will change, thus affecting the surface hydrophobicity of proteins [ 12 ]. ANS fluorescent probe method is the most commonly used method for the determination of protein surface hydrophobicity because it has the advantages of simple operation, fast operation, and a small amount of protein.…”

Section: Resultsmentioning

confidence: 99%

Effect of Steam Flash-Explosion on Physicochemical Properties and Structure of High-Temperature Denatured Defatted Rice Bran Protein Isolate

Wang

et al. 2023

Molecules

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The effects of Steam Flash-Explosion (SFE) on the physicochemical properties and molecular structure of high-temperature denatured defatted rice bran protein isolate (RBPI) were investigated. The mechanism of SFE treatment on high-temperature denatured defatted RBPI was revealed. The analysis of the physical and chemical properties of RBPI showed that the surface hydrophobicity, characteristic viscosity, and thermal stability of rice bran protein isolate were significantly affected by the pressure of saturated steam and pressure holding time. Under the conditions of 2.1 MPa and 210 s, the surface hydrophobicity index decreased significantly from 137.5 to 17.5, and the characteristic viscosity increased significantly. The peak temperature of denaturation decreases from 114.2 to 106.7 °C, and the enthalpy of denaturation decreases from 356.3 to 231.4 J/g. The higher structure (circular dichroic spectrum and endogenous fluorescence spectrum) of rice bran protein isolate was analyzed by volume rejection chromatography (SEC). The results showed that steam flash treatment could depolymerize and aggregate RBPI, and the relative molecular weight distribution changed greatly. The decrease in small molecules with poor solubility was accompanied by the increase in macromolecules (>550 kDa) soluble aggregates, which were the products of a Maillard reaction. The contents of free sulfhydryl and disulfide bonds in high-temperature rice bran meal protein isolate were significantly increased, which resulted in the increase in soluble aggregates containing disulfide bonds. Circular dichroism (CD) analysis showed that the α-helix content of the isolated protein was significantly decreased, the random curl content was increased, and the secondary structure of the isolated protein changed from order to disorder. The results of endogenous fluorescence spectroscopy showed that the high-temperature rice bran meal protein isolate was more extended, tryptophan was in a more hydrophilic microenvironment, the fluorescence intensity was reduced, and the tertiary structure was changed. In addition, the mean particle size and net surface charge of protein isolate increased in the aqueous solution, which was conducive to the development of the functional properties of the protein.

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Section: Resultsmentioning

confidence: 99%

Effect of Steam Flash-Explosion on Physicochemical Properties and Structure of High-Temperature Denatured Defatted Rice Bran Protein Isolate

Wang

et al. 2023

Molecules

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“…To evaluate the switching from an open to a closed form of productive MdxE-ligand complexes obtained with the sugars listed in Table 1 , changes in the protein surface hydrophobicity (H o ) of MdxE-ligand complexes using the fluorescent probe 8-anilinonaphthalene-1-sulfonate (ANS) were determined ( Figure 4 ). Since H o is governed by the slope generated as the fluorescence response changes upon binding interaction [ 47 , 48 ], increasing MdxE concentrations (2–14 µM) inside a linear range ( Figure S2 ) were tested using an excess of ligand (350 µM) at a fixed concentration of ANS (20 µM) to assure that MdxE was in its ligand-saturated state based on ITC studies ( Figure 3 ). First, a notable change in fluorescent response at λ max of 485 nm was observed with increasing MdxE-ligand concentrations at a constant ANS concentration (10 μM) ( Figure 4 A).…”

Section: Resultsmentioning

confidence: 99%

“…The changes in H 0 associated with the open-to-closed conformational changes of productive MdxE-ligand complexes were determined based on the method described previously [ 47 , 57 ], using a Nanodrop ND-3300 fluorospectrometer (Thermo Fisher, Waltham, MA, USA) controlled by ND-3300 v2.8.0 software with ANS as a fluorescence probe at an excitation wavelength of 365 nm and 400–600 nm as the range for emission scan. The fluorescent response was measured in RFU using buffer C for all solutions.…”

Section: Methodsmentioning

confidence: 99%

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Binding Specificity of a Novel Cyclo/Maltodextrin-Binding Protein and Its Role in the Cyclodextrin ABC Importer System from Thermoanaerobacterales

Aranda-Caraballo,

Saenz,

López-Zavala

et al. 2023

Molecules

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Extracellular synthesis of functional cyclodextrins (CDs) as intermediates of starch assimilation is a convenient microbial adaptation to sequester substrates, increase the half-life of the carbon source, carry bioactive compounds, and alleviate chemical toxicity through the formation of CD-guest complexes. Bacteria encoding the four steps of the carbohydrate metabolism pathway via cyclodextrins (CM-CD) actively internalize CDs across the microbial membrane via a putative type I ATP-dependent ABC sugar importer system, MdxEFG-(X/MsmX). While the first step of the CM-CD pathway encompasses extracellular starch-active cyclomaltodextrin glucanotransferases (CGTases) to synthesize linear dextrins and CDs, it is the ABC importer system in the second step that is the critical factor in determining which molecules from the CGTase activity will be internalized by the cell. Here, structure-function relationship studies of the cyclo⁄maltodextrin-binding protein MdxE of the MdxEFG-MsmX importer system from Thermoanaerobacter mathranii subsp. mathranii A3 are presented. Calorimetric and fluorescence studies of recombinant MdxE using linear dextrins and CDs showed that although MdxE binds linear dextrins and CDs with high affinity, the open-to-closed conformational change is solely observed after α- and β-CD binding, suggesting that the CM-CD pathway from Thermoanaerobacterales is exclusive for cellular internalization of these molecules. Structural analysis of MdxE coupled with docking simulations showed an overall architecture typically found in sugar-binding proteins (SBPs) that comprised two N- and C-domains linked by three small hinge regions, including the conserved aromatic triad Tyr193/Trp269/Trp378 in the C-domain and Phe87 in the N-domain involved in CD recognition and stabilization. Structural bioinformatic analysis of the entire MdxFG-MsmX importer system provided further insights into the binding, internalization, and delivery mechanisms of CDs. Hence, while the MdxE-CD complex couples to the permease subunits MdxFG to deliver the CD into the transmembrane channel, the dimerization of the cytoplasmatic promiscuous ATPase MsmX triggers active transport into the cytoplasm. This research provides the first results on a novel thermofunctional SBP and its role in the internalization of CDs in extremely thermophilic bacteria.

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“…(De la Cruz‐Torres et al . 2022). The sample was discharged into a phosphoric acid buffer with a final concentration of five protein samples (0.2–1.0 mg/mL, pH 7.0).…”

Section: Methodsmentioning

confidence: 99%

In vitro assessment of addition of soy protein isolate on milk protein digestion and conformational behaviour

Mo,

Yang,

Mao

et al. 2024

Int J of Dairy Tech

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Soy protein (SP) and milk protein are nutritionally complementary proteins, while they have different postprandial kinetic manifestations. In this study, different sources of proteins (casein, whey protein and SP isolate) were compounded in different concentrations and ratios. An in vitro dynamic gastrointestinal digestive system for children was used to study the digestive behaviour. For all the concentrations assessed, a consistently high level of small peptide release was demonstrated at 2.67 g/100 mL protein concentration, while the addition of 10% SP isolate would increase the release of small peptides. Overall, the study offers novel perspectives on children's products with diverse protein sources.

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Development of a rapid, high-sensitivity, low-cost fluorescence method for protein surface hydrophobicity determination using a Nanodrop fluorospectrometer

Cited by 8 publications

References 35 publications

Effect of Steam Flash-Explosion on Physicochemical Properties and Structure of High-Temperature Denatured Defatted Rice Bran Protein Isolate

Effect of Steam Flash-Explosion on Physicochemical Properties and Structure of High-Temperature Denatured Defatted Rice Bran Protein Isolate

Binding Specificity of a Novel Cyclo/Maltodextrin-Binding Protein and Its Role in the Cyclodextrin ABC Importer System from Thermoanaerobacterales

In vitro assessment of addition of soy protein isolate on milk protein digestion and conformational behaviour

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