Buried Sulfhydryl Groups Research Articles

Effect of ultrasound-assisted L-lysine treatment on pork meat quality and myofibrillar protein properties during postmortem aging.

This paper aimed to investigate the effects of ultrasound-assisted L-lysine treatment on meat quality and myofibrillar proteins (MPs) properties of pork longissimus dorsi during postmortem aging. The results revealed that the L-lysine (Lys) and/or ultrasound treatment significantly increased (p < 0.05) the water-holding capacity and tenderness of the pork during postmortem aging, while the ultrasound-assisted Lys treatment had the lowest cooking loss, pressurization loss, Warner-Bratzler shear force, and hardness. In addition, L-lysine and/or ultrasound treatment increased (p < 0.05) pH value, T21, and myofibrillar fragmentation index, while the ultrasound-assisted Lys treatment had the highest value. Meanwhile, the protein solubility was increased with Lys and/or ultrasound treatment during postmortem aging, and ultrasound-assisted Lys treatment had the highest solubility, reaching 88.19%, 92.98%, and 91.73% at 0, 1, and 3 days, respectively. The result of protein conformational characteristics showed that Lys and/or ultrasound treatment caused the unfolding of the α-helix structure, resulting in the exposure of more hydrophobic amino acids and buried sulfhydryl groups, ultimately enhancing MPs solubility. In summary, ultrasound-assisted Lys treatment altered the structure of MPs, resulting in the enhancement of the water-holding capacity and tenderness of the pork. PRACTICAL APPLICATION: This study showed that ultrasound-assisted L-lysine (Lys) treatment could enhance the water-holding capacity and tenderness of pork during postmortem aging. The results might provide a reference for the application of ultrasound-assisted Lys treatment on the improvement of pork meat quality. To facilitate practical applications in production, the development of medium and large-sized ultrasound equipment for conducting small-scale and pilot experiments is crucial for future research.

Combination of high-intensity ultrasound and hydrogen peroxide treatment suppresses thermal aggregation behaviour of myofibrillar protein in water

This study was aimed at evaluating the potential of high-intensity ultrasound (HIU, 450 W for 10 min) combined with hydrogen peroxide (H2O2) having various concentrations (0, 50, 100, 200, 400, 800 μmol/g protein) to inhibit the thermal aggregation behavior of myofibrillar proteins (MPs) in water. The results indicated that the addition of H2O2 interfered with the intermolecular sulfhydryl-disulfide interchange and inhibited the disulfide bond cross-linking. The H2O2-mediated conversion of cysteine to thiol derivatives appeared to be the primary mechanism of this effect. The HIU combined with H2O2, especially at the H2O2 concentration of 200 μmol/g, corresponded to a more significant inhibitory effect than that of only H2O2, which attributed to the dissociation of the filamentous myosin structure that led to an enhanced accessibility of the buried sulfhydryl groups. In conclusion, these findings provide direct evidence for the role of HIU combined with H2O2 in improving the thermal stability of MPs.

Heat-induced aggregation and gelation of proteins from edible honey bee brood (Apis mellifera) as a function of temperature and pH

Edible insects offer a huge potential as a new source of proteins for food and feed and as a promising functional agent. In this regard, our study aimed to evaluate the effect of pH and temperature on aggregation and gelation of honey bee brood (Apis mellifera). Aggregation of soluble proteins was dependent on temperature and pH value and the highest coagulation was reached at 85 °C at pH 5 and 7 (73.7 and 68.4%, respectively). Changes in protein surface hydrophobicity, net charge and amount of available and buried sulfhydryl groups demonstrated the contribution of either covalent or non-covalent intermolecular interactions resulted in aggregation at various pH. The least gelation concentration of raw powder from honey bee brood varied from 5 to 11% on raw powder basis at pH 7 and 3, respectively. Moreover, the effect of pH on rheological and textural properties, as well as gel microstructure from honey bee brood is discussed. Therefore, freeze dried raw powder of honey bee brood has rich nutritional value and demonstrated gelation concentration comparable to conventional protein sources that reveal its potential as an ingredient for insect-based gel product.

The mechanism of high pressure-induced gels of rabbit myosin

Abstract Rabbit myosin (20 mg/mL of 0.6 M NaCl, pH 6.5) was subjected to various pressures (100–400 MPa) at 20 °C for 10 min to investigate pressure-induced gel mechanism by measurements of protein surface hydrophobicity, sulfhydryl contents, molecular size by native polyacrylamide gel electrophoresis, ultra-structure using SEM, and viscoelastic properties by dynamic rheology. Surface hydrophobicity and sulfhydryl groups had little increase at 100 and 200 MPa, but had significant increase at 300 and 400 MPa. Ultra-structure revealed that gels below 200 MPa were filament structure with many small cavities, while gels upon 300 MPa were globular aggregates with big cavities. Dynamic rheological measurements indicated that gels were formed at 400 MPa. Mechanism of myosin gel formation in pressure treatment was that myosin was unfolded, following the exposure of hydrophobic and buried sulfhydryl groups, then was denatured (electrophoresis showed the heavy chain became very weak) and associated to form gels. Industrial relevance In recent years, high pressure treatment of food has received interest as an alternative to heat treatment for gel formation. Gel-type meat products of high pressure processing are still not commercialized. This is basically due to lack of information about high pressure-induced gel. Myosin is the protein responsible for the gelling capacity of muscle systems. The processing characteristics of gels treated by high pressure were improved. The results of this experiment showed that myosin denatured and formed gels at pressure 400 MPa holding for 10 min. This information will help industry to adopt high pressure to produce meat products.

The use of β-glucan as a partial salt replacer in high pressure processed chicken breast meat

The effect of various ingredients such as sodium chloride (NaCl), sodium tripolyphosphate (STPP) and β-glucan (BG) on the biochemical properties of chicken breast proteins during temperature assisted high pressure processing was studied. Total protein solubility revealed that 600MPa pressure and 40oC are critical for the denaturation of proteins in STPP samples. Increase in reactive sulfhydryl groups with pressure indicate the exposure of buried sulfhydryl groups. Hydrophobicity and sulfhydryl contents revealed that hydrophobic interaction and disulphide bond formation are responsible for gel formation. The study revealed that 40oC and 400/600MPa pressure is optimum for high pressure processing of chicken breast meat. Addition of β-glucan with reduced NaCl and in the absence of sodium tripolyphosphate could produce gels with similar properties to those with 2.5% NaCl addition. Hence it is proposed that β-glucan can be used to reduce NaCl content of chicken products produced by temperature assisted high pressure processing.

Effect of heat-treatment on the physico-chemical properties of egg white proteins: A kinetic study

In this study, kinetic models were established to describe the heat-induced changes in selected physico-chemical properties of egg white proteins. These changes were studied in a temperature range of 50–85 °C and two pH levels were selected, to mimic the pH of fresh (pH 7.6) and aged egg white (pH 8.8). The heat-induced decrease of residual denaturation enthalpy and amount of buried sulfhydryl groups and the increase in surface hydrophobicity could be described at both pH levels with a first order fractional conversion model, while this model could only be applied at pH 7.6 to describe the loss of protein solubility and increase in turbidity due to heat-treatment. A second order model could be used to characterize the heat-induced decrease of total sulfhydryl content. The rate constants to describe the changes in denaturation enthalpy and buried sulfhydryl content were more sensitive to temperature increase than the other properties studied, as indicated by a higher activation energy. These kinetic data can be used to predict heat-induced changes in related functional properties of egg white proteins.

Changes in Sulfhydryl Content of Egg White Proteins Due to Heat and Pressure Treatment

The sulfhydryl (SH) content of egg white proteins (10% v/v or 9.64 mg of protein/mL) after heat (50-85 degrees C) and combined heat- and high-pressure treatments (100-700 MPa, 10-60 degrees C) was determined using 5',5-dithiobis (2-nitrobenzoic acid) (DTNB), both for the soluble fraction and the total protein fraction. Only irreversible changes were taken into account. Both physical treatments were performed at two pH levels: pH 7.6, corresponding to the pH of fresh egg white, and pH 8.8, corresponding to that of aged egg white. Both heat and combined heat- and high-pressure treatment resulted in an exposure of buried SH groups. These exposed SH groups were involved in the formation of disulfide bond stabilized protein aggregates, as shown by gel electrophoresis. Under severe processing conditions (above 70 degrees C at atmospheric pressure or above 500-600 MPa, depending on the temperature applied), a decrease in total SH content could be observed, probably due to the formation of disulfide bonds by oxidation, especially at alkaline pH when the thiolate anion was more reactive. The high degree of exposure of sulfhydryl groups, and subsequent oxidation and sulfhydryl-disulfide bond exchange reactions resulting in soluble aggregates, can explain why pressure-induced egg white gels are softer and more elastic than heat-induced ones. When pressure treatment was performed at low temperatures (e.g., 10 degrees C), a lower pressure was required to induce similar changes in the sulfhydryl content, as compared to higher temperatures (e.g., 25 degrees C), indicating an antagonistic effect between pressure and temperature in the domain studied (10-60 degrees C, 100-700 MPa). Treatment conditions resulting in extensive protein insolubilization were accompanied by a transfer of free sulfhydryl groups from the soluble to the insoluble protein fraction. These SH groups were mainly accessible to DTNB.

Dimerization occurs during the reversible acid inactivation of 2-oxo-4-hydroxyglutarate aldolase from Escherichia coli

Exposure of Escherichia coli 2-oxo-4-hydroxyglutarate aldolase (4-hydroxy-2-oxoglutarate glyoxylate-lyase, EC 4.1.3.16) (molecular weight = 63000) to phosphoric acid at pH 1.6 for 10 min at 4δC causes 95% or greater inactivation. No significant effect on the rate or extent of inactivation is caused by varied aldolase concentrations or the presence of exogenous proteins. Chloride ion (50–100 mM) or 10 mM 2-oxo-4-hydroxyglutarate markedly decreases both the rate and extent of inactivation; good protection is also afforded by 10 mM pyruvate, glyoxylate, glyoxal, 2-oxoglutarate or 2-oxobutyrate. Whereas native aldolase has two free and three buried sulfhydryl groups, all five are exposed in the acid-inactivated enzyme and the molecular weight of this species at pH 1.6 is 126000. Ultraviolet absorbance difference spectra, circular dichroism spectra and ultracentrifugation studies establish that the inactivation process is characterized by an alteration of secondary and tertiary structure as well as an aggregation to a dimer of the native molecule. Reactivation of enzyme activity to 60–80% of the original level is seen within 20 min at pH 6 to 8; examination of inactivation/reactivation as a function of pH indicates that these two processes occur via kinetically distinct pathways. Native and reactivated enzymes are identical in molecular weight, sulfhydryl titer, K m and α-helix content.

Modification of bovine heart mitochondrial transhydrogenase with tetranitromethane

Modification of pyridine dinucleotide transhydrogenase with tetranitromethane resulted in inhibition of its activity. Development of a membrane potential in submitochondrial particles during the reduction of 3-acetylpyridine adenine dinucleotide (AcPyAD +) by NADPH decreased to nearly the same extent as the transhydrogenase rate on tetranitromethane treatment of the membrane. Kinetics of the inactivation of homogeneous transhydrogenase and the enzyme reconstituted into phosphatidylcholine liposomes indicate that a single essential residue was modified per active monomer. NADP +, NADPH and NADH gave substantial protection against tetranitromethane inactivation of both the nonenergy-linked and energy-linked transhydrogenase reactions of submitochondrial particles and the NADPH → AcPyAD + reaction of reconstituted enzyme. NAD + had no effect on inactivation. Tetranitromethane modification of reconstituted transhydrogenase resulted in a decrease in the rate of coupled H + translocation that was comparable to the decrease in the rate of NADPH → AcPyAD + transhydrogenation. It is concluded that tetranitromethane modification controls the H + translocation process solely through its effect on catalytic activity, rather than through alteration of a separate H +-binding domain. Nitrotyrosine was not found in tetranitromethane-treated transhydrogenase. Both 5,5′-dithiobis(2-nitrobenzoate)-accessible and buried sulfhydryl groups were modified with tetranitromethane. NADH and NADPH prevented sulfhydryl reactivity toward tetranitromethane. These data indicate that the inhibition seen with tetranitromethane results from the modification of a cysteine residue.

Essential sulfhydryl groups and the light-dependent transhydrogenase system of Rhodopseudomonas spheroides: localization of substrate binding sites and evidence for masked or buried sulfhydryl groups in the peripheral protein factor.

The light-dependent transhydrogenase system of Rhodopseudomonas spheroides which consists of a peripheral protein factor and a membrane-bound component contains essential sulfhydryl groups that are sensitive to p-hydroxymercuribenzoic acid (PMB). There are two types of sulfhydryl groups required for light-dependent (LD) transhydrogenation. One type is associated with the membrane-bound component and participates in or influences the binding of one of the substrates, NADH. A second type is associated with the peripheral protein factor and is not involved with the binding of the substrates. These sulfhydryl groups are masked or buried in the protein and are only exposed upon treatment of the peripheral protein factor with urea or trypsin. The peripheral protein factor may be an integral part of the transhydrogenase complex or may be required for the energization process. This factor appears to play an important role in the activation and control of LD-transhydrogenation.

A spin label study of erythrocyte membranes during simulation of freezing.

Human erythrocytes were labeled with stearic acid spin labels, and no change was detected in membrane fluidity under hyperosmotic stress, going from isotonicity to about 3000 mOsm. Intact erythrocytes labeled with an androstane spin label and submitted to simulation of freezing show the onset of irreversible structural breakdown occurring in a saline solution at 2,000 mOsm. Ghosts labeled with maleimide spin label (4-maleimide-2,2,6,6-tetramethylpiperidinooxyl) when submitted to solutions of increasing osmolalities (pH 7.4), exhibit protein conformational changes that are irreversible after a simulated freeze-thaw cycle. After sonication of maleimide spin-labeled ghosts, membrane buried sulfhydryl groups become exposed. Such preparations showed behavior similar to the unsonicated when in saline hyperosmolal medium (pH 7.4). Such results suggest the ionic strength of the medium as the determining factor of the detected conformational changes. Maleimide spin-labeled ghosts in 300 mOsm saline solution (pH 7.4) were treated with ascorbic acid (spin destruction of nitroxides), and the kinetic analysis indicates that 65% of the labeled sites are located at the external interface of the membrane or in hydrophilic channels. Deformation and rearrangements of membrane components in solutions of increasing osmolalities apparently are related to protein conformational changes, on the outside surface of erythrocyte membranes, with a significant amount being structurally dissociated of lipids.

Effects of cyclic nucleotides on the conformational states of the α core of the cyclic amp receptor protein

The α core gragment produced by limited proteolysis contains the cyclic AMP binding domain and the two buried sulfhydryl groups of the cyclic AMP receptor protein. The buried sulfhydryl groups of the α core react with 5,5′-dithio-bis(2-nitro-benzoic acid) after denaturation by 3 M urea or digestion with subtilisin. The rate of sulfhydryl modification in the presence of 3 M urea or subtilisin is markedly decreased in the presence of cyclic nucleotides which are proposed to tighten the conformation of the α core. Incubation of the α core in 3 M urea or dithionitrobenzoic acid does not affect cyclic AMP binding while dithionitrobenzoic acid plus 3 M urea inhibits cyclic AMP binding suggesting a role for the buried sulfhydryls in cyclic AMP binding or their proximity to the cyclic AMP binding domain of the α core. The data are consistent with a ligand-induced conformational change in the α region of the native cyclic AMP receptor protein that is required for DNA binding.

Studies on the characterization of the sodium—potassium transport adenosinetriphosphatase: XIII. On the organization and role of phospholipids in the purified enzyme

Spin-labeling with 7- and 12-nitroxide-labeled stearic acids and high- and low-angle X-ray diffraction provide strong evidence that the phospholipid in the purified (sodium + potassium)-activated adenosinetriphosphatase from the rectal gland of Squalus acanthias possesses bilayer character. Treatment with phospholipase A produced a change in the ESR spectrum of the stearate-12-nitroxide-labeled enzyme compatible with that seen with erythrocyte and nonmyelinated nerve axon plasma membranes and may be explained by a condensation of the bilayer or the absorption of the spin label in more hydrophilic membrane sites. Phospholipase A treatment also produced a change in the ESR spectrum of spinlabeled NEM-treated enzyme which suggests an increased exposure of buried sulfhydryl groups to a more polar environment. It is suggested that inactivation of the NaK ATPase by phospholipase A digestion may be due to a conformational change in the protein as a result of destabilization of the protein associated with disruption of the phospholipid bilayer.

N-acylsarcosines as inhibitors of respiration and glycolysis and glycolytic enzymes

Sodium- N-lauroyl sarcosinate and sodium- N-palmitoyl sarcosinate inhibit the respiration and anaerobic glycolysis of isolated mouse diaphragm. Rabbit heart glucokinase and rabbit muscle fructose-1,6-diphosphate aldolase are also inhibited by SLS and SPS, which appear to act as denaturing agents. In concentrations ranging from 1 × 10 −2 m to 3 × 10 −2 m for SLS and 2.5 × 10 −5 m to 2.5 × 10 −4 m for SPS both compounds cause progressive unfolding of rabbit muscle aldolase, as evidenced by the exposure of all the buried sulfhydryl groups; this process is associated with loss of enzymatic activity.