Exposure Of Tryptophan Residues Research Articles

High-moisture extrusion of yeast-pea protein: Effects of different formulations on the fibrous structure formation

The demand of meat analogues (MAs) is consistently increasing. The protein materials for MAs are primarily soy, pea, and wheat protein which can not completely meet the growing demand. Hence, this study is focused on the preparation of MAs with up to 50 % yeast protein (YP) instead of pea protein isolate (PPI). In the present study, 0 %, 10 %, 30 %, and 50 % YP powder in dry matter basis were combined with PPI; then the mixtures were used to prepare MAs with fibrous structures using high-moisture extrusion (55 % moisture). The involvement of YP significantly enhanced the hardness of MAs (P < 0.05). The optical and microstructural images illustrated that when YP ratio reached 30 %, obvious fibrous structures still were observed in MAs. Furthermore, MAs containing YP became whiter, which is conducive to reprocessing. With an increase in YP, the bound water content, sheet structures, and exposure of tryptophan residues in MAs increased, whereas the free water content, β-turn, and random coil structures decreased. Analysis of thermal and rheological behaviors indicated that YP lowered the denaturation temperature of MAs and the viscosity of protein dispersions, which was related to the formation of protein aggregates. Overall, YP can be used to prepare MAs and regulate the fibrous structure in MAs by acting on protein conformations.

Mechanism of differences in characteristics of thick/thin egg whites during storage: Physicochemical, functional and molecular structure characteristics analysis

This study systematically analyzed and compared thechanges of physicochemical, functional and molecular structural characteristics between thick egg white (KEW) and thin egg white (NEW) during storage. Analysis of physicochemical properties showed that moisture content decreased significantly with the increase of pH during storage. KEW was gradually thinning, while NEW was closer to Newtonian fluid. Functional properties indicated that KEW thermal gel was gradually hard and brittle with the properties of NEW. KEW had better emulsifying property than NEW, and NEW had superior foaming ability. The α-helix and β-sheet in the FT-IR spectrum showed a downward trend, revealing secondary structure changed from order to disorder. Enhancement of fluorescence intensity indicated the structural unfolding and exposure of tryptophan residues. SDS-PAGE proved that OVO might be related to the difference between KEW and NEW characteristics. This study provided new idea and reference value for egg storage and diversified utilization of egg white.

Effect of high pressure processing on fibrinolytic activity of fruit bromelain in vivo

AbstractAiming at exploring how high pressure processing (HPP) affects the fibrinolytic activity of fruit bromelain (FB) in vivo, the obtained FB was subjected to HPP treatment (300 MPa, 20°C for 20 min) before advanced structure was determined; then six groups of rats were fed with various dosages of enzymes (0.5–1.5 mL/kg·bw) and six target parameters of blood plasma were analyzed. Structural measurements revealed that, by comparison with FB, HPP treated FB (HPP‐FB) showed an increase (7.67%) in α‐helix and its fluorescence intensities were increased by 42.5%, 78.7% at the excitation wavelength of 295 nm, 280 nm, respectively. As to the fibrinolytic activity, compared with the blank control, activated partial thromboplastin time (APTT), prothrombin time (PT) and tissue‐type plasminogen activator (t‐PA) of Group 8 (highest HPP‐FB dosage) were markedly increased (p &lt; .05) by 18.40%, 16.57%, and 10.68% separately, while fibrinogen (FIB) was significantly decreased (p &lt; .05) by 13.88%. Besides, compared with the positive control, the highest HPP‐FB dosage performed almost the same function as aspirin. The results indicated that, compared with FB, the fibrinolytic activity of HPP‐FB was obviously increased which might be attributed to the increase of α‐helix and the exposure of tryptophan residues and tyrosine residues.Practical applicationsThe results in this research suggested that HPP treatment could effectively enhance the fibrinolytic activity of fruit bromelain in vivo and increase the added‐value as well as market competitiveness of pineapple‐derived products. HPP‐FB could potentially be used in the development of a new functional food that can prevent thrombosis.

Solvent-mediated pressure-treated bixin-casein complexation for targeted color delivery.

Carryover color in the whey fluid is one of the major challenges faced by the cheese manufacturing industry. In this study, we describe a solvent-mediated high-pressure process to complexate bixin and casein micelles as a novel strategy for color delivery. High pressures (120 and 240 MPa) and added ethanol resulted in change in casein hydrophobicity by exposure of tryptophan residues, as confirmed by spectroscopic methods. The incorporation of bixin resulted in a marked quenching of the fluorescence peak associated with tryptophan. A simulated coagulation study has shown that pressure-treated complexes resulted in whey powder with significantly lower a* values than unbleached whey, whereas no significant differences were observed for b* values. The results suggest that complexes can produce curds with a color similar to that using commercial annatto powder and whey powder with equal or superior color quality than obtained with chemical bleaching.

Bovine lactoferrin binds oleic acid to form an anti-tumor complex similar to HAMLET

α-Lactalbumin (α-LA) can bind oleic acid (OA) to form HAMLET-like complexes, which exhibited highly selective anti-tumor activity in vitro and in vivo. Considering the structural similarity to α-LA, we conjectured that lactoferrin (LF) could also bind OA to obtain a complex with anti-tumor activity. In this study, LF–OA was prepared and its activity and structural changes were compared with α-LA–OA. The anti-tumor activity was evaluated by methylene blue assay, while the apoptosis mechanism was analyzed using flow cytometry and Western blot. Structural changes of LF–OA were measured by fluorescence spectroscopy and circular dichroism. The interactions of OA with LF and α-LA were evaluated by isothermal titration calorimetry (ITC). LF–OA was obtained by heat-treatment at pH8.0 with LD50 of 4.88, 4.95 and 4.62μM for HepG2, HT29, and MCF-7 cells, respectively, all of which were 10 times higher than those of α-LA–OA. Similar to HAMLET, LF–OA induced apoptosis in tumor cells through both death receptor- and mitochondrial-mediated pathways. Exposure of tryptophan residues and the hydrophobic regions as well as the loss of tertiary structure were observed in LF–OA. Besides these similarities, LF showed different secondary structure changes when compared with α-LA, with a decrease of α-helix and β-turn and an increase of β-sheet and random coil. ITC results showed that there was a higher binding number of OA to LF than to α-LA, while both of the proteins interacted with OA through van der Waals forces and hydrogen bonds. This study provides a theoretical basis for further exploration of protein–OA complexes.

Brief heat treatment causes a structural change and enhances cytotoxicity of the Escherichia coli α-hemolysin

α-Hemolysin (HLY) is an important virulence factor for uropathogenic Escherichia coli. HLY is a member of the RTX family of exotoxins secreted by a number of Gram-negative bacteria. Recently, it was reported that a related RTX toxin, the Mannheimia haemolytica leukotoxin, exhibits increased cytotoxicity following brief heat treatment. In this article, we show that brief heat treatment (1 min at 100°C) increases cytotoxicity of HLY for human bladder cells, kidney epithelial cells (A498) and neutrophils. Heat treatment also increased hemolysis of human red blood cells (RBCs). Furthermore, heat treatment of previously inactived HLY restored its cytotoxicity. Heat-activated and native HLY both required glycophorin A to lyse RBCs. Native and heat-activated HLY appeared to bind equally well to the surface of A498 cells; although, Western blot analyses demonstrated binding to different proteins on the surface. Confocal microscopy revealed that heat-activated HLY bound more extensively to internal structures of permeabilized A498 cells than did native HLY. Several lines of spectroscopic evidence demonstrate irreversible changes in the structure of heat activated compared to native HLY. We show changes in secondary structure, increased exposure of tryptophan residues to the aqueous environment, an increase in molecular dimension and an increase in hydrophobic surface area. These properties are among the most common characteristics described for the molten globule state, first identified as an intermediate in protein folding. We hypothesize that brief heat treatment of HLY causes a conformational change leading to significant differences in protein–protein interactions that result in increased cytotoxicity for target cells.

Raman spectroscopic study of deamidated food proteins

Three food protein products, soy and whey protein isolates and spray-dried egg white powder, were deamidated to various levels by 0.04 N hydrochloric acid. The extents of deamidation were determined using an ammonia electrode. Raman spectra of the modified proteins were obtained and analyzed. A new C O stretching vibration band was observed at 1780 cm −1, attributed to γ-carboxyl groups of aspartic and glutamic acids. Calibration curves were constructed by plotting the intensity ratio of the 1780 cm −1 band to the 1003 cm −1 phenylalanine stretching band (as an internal standard) against the extent of deamidation. Linear fits were obtained with high correlation coefficients r > 0.987. Effect of deamidation on the conformation of these proteins was also studied by monitoring changes in Raman spectral characteristics, including a transition from ordered to disordered structures, exposure of tryptophan residues from a buried, hydrophobic microenvironment, and probable conformational changes of the aliphatic residues.

Partially Folded Conformations of Bovine Liver Glutamate Dehydrogenase Induced by Mild Acidic Conditions

The acid-induced unfolding of bovine liver glutamate dehydrogenase (GDH) was studied using various spectroscopic methods such as far- and near-UV circular dichroism (CD), intrinsic and 1-anilino naphthalene-8-sulphonate (ANS) extrinsic fluorescence spectroscopy, light scattering and fluorescence quenching in 20 mM mixed buffer at various pHs. CD spectra show that at pH 3.5, GDH retains its secondary structure substantially, whereas its tertiary structure content is reduced considerably. Intrinsic fluorescence of GDH and ANS binding suggest that, at pH 3.5, the hydrophobic surface of enzyme is more exposed in comparison to the native form. Acrylamide quenching indicates more exposure of tryptophan residues of enzyme at pH 3.5 in comparison to pH 7.5. Another partially unfolded intermediate was detected at pH 5.0, which with its ANS binding capacity lies between the pH 3.5 intermediate and the native form of the enzyme. Gel filtration results revealed that the enzyme at pH 3.5 is dissociated into trimeric species whereas it exists as hexamer at pH 7.5 and 5.0. All the data taken together suggest the existence of two partially unfolded states of GDH at moderate acidic pHs which may be considered as molten and pre-molten globule-like states.

Study of Succinylated Food Proteins by Raman Spectroscopy

Three food protein products, soy protein isolates, spray-dried egg white, and whey protein isolates, were chemically modified to varying levels with succinic anhydride, and the extent of modification of these proteins were determined by standard wet chemistry methods. Raman spectra (500-2000 cm(-1)) of the modified proteins were obtained. New C=O stretching vibrations were observed at 1420 and 1737 cm(-1) and were attributed to the carboxylate (COO-) and ester carbonyl (RCOO-) groups, respectively, which were appended to the proteins during succinylation. Two series of calibration curves were obtained by plotting the intensity ratio of the 1420 and 1737 cm(-1) to 1003 cm(-1) phenylalanine stretching band (used as an internal standard) against the extent of substituted epsilon-amino (and alpha-amino) groups and aliphatic hydroxyl groups, respectively. Linear fits were obtained with correlation coefficient r > 0.988. The Raman spectral data were also analyzed to study the effect of succinylation on the conformation of the three proteins. Some conformational changes were observed, including a transition from ordered to disordered structures, an exposure of tryptophan residues from a buried, hydrophobic microenvironment, and probably conformational shift of cystine residues.

Preparation of antimicrobial reduced lysozyme compatible in food applications.

The structural and antimicrobial functions of lysozyme reduced with food-compatible reducing agents-cysteine (Cys) and glutathione (GSH)-were investigated. The disulfide bonds were partially reduced by thiol-disulfide exchange reactions under heat-induced denaturing conditions from 55 to 90 degrees C. The results showed that treatment of lysozyme with Cys and GSH resulted in the introduction of new half-cystine residues (2-3 residues/mol of protein). The released SH groups, in turn, rendered the lysozyme molecule more flexible, being accompanied by a dramatic increase in the surface hydrophobicity and exposure of tryptophan residues. As a consequence, the resulting reduced lysozymes were more capable of binding to lipopolysaccharides (LPS) and permeabilizing the bacterial outer membrane, as evidenced by the liposome leakage experiment, than were native or heated lysozyme. Both reduced lysozymes displayed significantly higher antimicrobial activity than native or heated lysozyme against Salmonella enteritidis (SE) in sodium phosphate buffer (10 mM, pH 7.2) at 30 degrees C for 1 h. Their minimal inhibitory concentrations (MICs) against the tested bacteria were about 150- and 25-fold lower than their respective MICs of native or heated lysozyme. The results suggest that partially reduced lysozyme could be used as a potential antimicrobial agent for prevention of SE attack.

A Structural Phase of Heat-Denatured Lysozyme with Novel Antimicrobial Action

The structure and antimicrobial function of hen egg white lysozyme was investigated by means of thermal denaturation at 80 °C (pH 7.2), which leads to irreversible denaturation. With an increase in the heating time (up to 30 min) of lysozyme, the soluble fraction showed progressive decrease in its enzyme activity that coincided with the formation of a slower migrating band on the acid PAGE. Fluorescence spectra revealed that, as the extent of denaturation increases, the surface hydrophobicity and the exposure of tryptophan residues were greatly promoted. In parallel to these conformational changes of lysozyme there has been consistent increase in its antimicrobial activities against Gram-negative bacteria, with no detrimental effect on its inherent action to Gram-positive bacteria. Interestingly, lysozyme heated for 20 min, devoid of enzyme activity (HDLz), killed Escherichia coli K12 in a dose-dependent manner, while its bactericidal activity to Staphylococcus aureus was almost similar to that of the native lysozyme. The binding capacity of HDLz to membrane fractions of E. coli K12 was greatly promoted, particularly to the inner membrane, as determined by ELISA. The HDLz permeabilized liposomal membranes made from E. coli phospholipids, as demonstrated by calcein efflux, in a protein concentration-dependent manner. Good correlations between the degree of heat inactivation of lysozyme (or dimerization), increased hydrophobicity, and enhanced bactericidal activity against Gram-negative E. coli K12 were observed. The results of this study, first of all, suggest that susceptibility of Gram-negative or even Gram-positive bacteria to lysozyme is independent of enzymatic activity. It is likely that denatured lysozyme, e.g., the dimeric form, has an intrinsic structural motif which is generally lethal to the bacteria through membrane perturbation. Keywords: Lysozyme; denaturation; conformational changes; antimicrobial action; membrane interaction; liposome

Fluorescence studies of extracellular hemoglobin of Glossoscolex paulistus obtained by gel filtration

Chromatography in Sephadex G-200 of extracellular hemoglobin of the giant worm Glossoscolex paulistus presents a unique band at pH 7.0 and several bands at pH 9.0 as a result of alkaline dissociation. SDS-polyacrylamide gel electrophoresis (PAGE) of the intact protein obtained at pH 7.0 shows the existence of five different bands with molecular weights of 12 ± 1, 26 ± 3, 28 ± 2, 34 ± 1 and 53 ± 1 kDa. In the presence of β-mercaptoethanol, six distinct bands are obtained with molecular weights 13 ± 1, 14.8 ± 0.3, 15.8 ± 0.2, 16.6 ± 0.2, 35.1 ± 0.3 and 40.5 ± 0.5 kDa. Reduction of disulfide bonds of a trimer of molecular weight 53 kDa leads to the appearance of monomeric subunits of 14.8, 15.8 and 16.6 kDa. The molecular weights obtained from chromatography in Sephadex G-200 at pH 9.0 are different from those from SDS-PAGE. An intense band at 110 ± 12 kDa due to the tertiary complex of two disulfide-linked trimers and monomer chain (trimer + monomer), (abcd)2, is observed in addition to a small amount of high molecular weight fraction in the exclusion volume, as well as poorly resolved trimer (42 ± 2 kDa) and monomer (15 ± 2 kDa) bands. Fluorescence quantum yields of different fractions obtained in the chromatography as well as extinction coefficients at 280 and 415 nm were estimated and compared with human hemoglobin. The fluorescence data presented are consistent with the high content of aromatic residues in the G. paulistus hemoglobin. The increase in the fluorescence quantum yields upon both alkalinization and dissociation were correlated with the exposure of tryptophan residues to the solvent. The intact native protein has a quantum yield of 0.25% at pH 7.0, assigning 14% to tryptophan solution at pH 7.0 and increasing to 0.8% at pH 9.0 upon dissociation. The monomer has a fluorescence quantum yield of 0.8% at pH 7.0, increasing to 2.1% at pH 9.0. The maximum emission wavelength of the intact protein (327 nm) is consistent with tryptophan residues buried, quenched and becoming more exposed and more fluorescent upon dissociation into smaller subunits (332-335 nm). In the monomer the tryptophan residues remain buried inside the protein molecule at pH 9.0.

Reversible and irreversible modifications of beta-lactoglobulin upon exposure to heat.

Modifications in the exposure to the solvent of hydrophobic residues, changes in their organization into surface hydrophobic patches, and alterations in the dimerization equilibrium of beta-lactoglobulin upon thermal treatment at neutral pH were studied. Exposure of tryptophan residues was temperature dependent and was essentially completed on the time scale of seconds. Reorganization of generic hydrophobic protein patches on the protein surface was monitored through binding of 1,8-anilinonaphthalenesulfonate, and was much slower than changes in tryptophan exposure. Different phases in surface hydrophobicity changes were related to the swelling and the subsequent collapse of the protein, which formed a metastable swollen intermediate. Heat treatment of beta-lactoglobulin also resulted in the formation of soluble oligomeric aggregates. The aggregation process was studied as a function of temperature, demonstrating that (i) dimer dissociation was a necessary step in a sequential polymerization mechanism and (ii) cohesion of hydrophobic patches was the major driving force for aggregation.

Differential Exposure of Tryptophan Residues in the Red and Far-Red Light Absorbing Forms of Phytochrome, as Revealed by Chemical Modification

Differential Exposure of Tryptophan Residues in the Red and Far-Red Light Absorbing Forms of Phytochrome, as Revealed by Chemical Modification

Distribution analysis of membrane penetration of proteins by depth-dependent fluorescence quenching

A new approach is presented to evaluate the depth-dependent quenching of the fluorescence of membrane-bound probes and integral proteins. By utilizing at least three quenchers of known and distinctly different depths, the following parameters can be recovered: most probable depth of the probe; dispersion of the depth distribution, which will depend on the size of probe and fluctuations in its position; and quenching efficiency, which is related to the exposure of a particular fluorophore to the lipid phase. The exposure of tryptophan residues in integral proteins can be quantitatively determined with respect to the model compound (tryptophan octyl ester). The proposed method was applied to the investigation of membrane complexes of the bee venom melittin and cytochrome b5.

Enzymatic deglycosylation of human thyroglobulin: fluorescence studies.

The interaction between the carbohydrate and the amino acid residues in human thyroglobulin has been studied. Previous reports showed that the removal of the two terminal carbohydrates of the complex chains leads to an increase in thyroglobulin binding to thyroid membranes. In our study, after enzymatic release with glycosidases of the sugar moieties from thyroglobulin, a time-dependent decrease in tryptophan fluorescence has been observed. This decrease was also associated with a shift in the emission peak from 335 to 340 nm. The strong quenching of tryptophan emission was also accompanied by a decrease in the exposure of tryptophan residues, as shown by a Stern-Volmer analysis with the neutral quencher acrylamide. These data, together with the increase in fluorescence of the dansylated deglycosylated thyroglobulin, strongly suggest that a significant conformational change of thyroglobulin follows the deglycosylation of the protein.

Hydrogen Ion-induced Reversible Inactivation and Dissociation of Formyltetrahydrofolate Synthetase

Abstract Formyltetrahydrofolate synthetase undergoes inactivation at pH values below 7. Inactivation is accompanied by both the dissociation of the tetramer into monomers and the exposure of aromatic amino acids to the solvent. The rate of inactivation is a function of pH. This rate and the rate of exposure of tryptophan residues to the aqueous environment follow first order kinetics. After prolonged incubation under acidic conditions the monomers are still capable of reassociating to form tetramers but the tetramers produced have a reduced catalytic activity. The alterations in enzyme structure and function which occur during inactivation are reversed following an elevation of pH. The return of enzymic activity is accompanied by both the reassociation of monomers and the burying of exposed tryptophan residues. The rate of reactivation and the rate of burying of tryptophan follow first order kinetics. The first order rate constant for reactivation varies as a function of temperature, and the heat of activation calculated from the linear Arrhenius plot is 20.1 kcal per mole. The free energy of activation and entropy of activation at 25° have values of 21 kcal per mole and -3 e.u., respectively. A mechanism in which the rate-limiting step for reactivation is a conformational change of the monomer that occurs prior to reassociation is consistent with the experimental observations. Both the rate and extent of reactivation increase as the pH is raised from 6 to 7.9. The data suggest that reactivation is dependent on the deprotonation of histidine residues. The presence of an optimum concentration of sulfate enhances both the rate and extent of reactivation at pH values between 6 and 7. The presence of a thiol compound during inactivation and reactivation is required to achieve a maximum restoration of enzymic activity. It is suggested that the monomer is unstable in the absence of a thiol and is converted to other monomer forms possibly as a result of oxidation of sulfhydryl groups. These monomer forms are not converted to the active tetramer under the conditions used. The extent of reactivation is dependent upon protein concentration. The optimum protein concentration depends on the pH at which reactivation is performed. As the pH of reactivation is lowered toward pH 6, the concentration of protein required to achieve reactivation increases. These results are explained by assuming that an equilibrium exists between monomer and tetramer, and that the production of the monomer which is capable of reassociating to form the active tetramer results from a conformational change caused by a protonation of a residue with a pKa of about 6.0.

Photooxidation of glucoamylase I from aspergillus niger

Photooxidation of glucoamylase I in the presence of methylene blue results in the abolition of the enzymic activity. The process is pH- dependent, being more rapid at high pH. Loss of activity appears to be associated with destruction of tryptophan residues. The pH- dependence of the process is interpreted in terms of a conformational change undergone by the enzyme resulting in exposure of tryptophan residues which may be essential either directly for the activity or for the maintenance of the active three-dimensional structure.