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PubMed2004

Turkey carcass chilling and protein denaturation in the development of pale, soft, and exudative meat.

Pale, soft, and exudative (PSE) meat is a growing problem in the turkey industry and has been associated with processing conditions such as slow carcass chilling. The development of PSE meat is caused by protein denaturation resulting from a rapid rate of pH decline early postmortem (PM) while carcass temperatures are still elevated. This research was conducted to determine the relationship of slow chilling to protein denaturation and PSE development. A total of 48 toms were conventionally processed in 2 trials at 22.5 wk of age, and chilled at 0, 10, 20, or 30 degrees C for either 45 or 90 min before deboning (at 60 or 105 min PM). Temperature and pH of the breast muscle was recorded at 15 min PM, at the time of deboning (60 or 90 min PM), and at 24 h PM. Color was determined at deboning and again at 24 h PM. Gel strength, cook loss, expressible moisture, total protein solubility, and bound phosphorylase quantities were determined on the fillets at 24 h PM. There was no difference in carcass temperature at 15 min PM, but by 105 min PM each temperature treatment was significantly different, with the carcasses chilled at 0 and 10 degrees C having the lowest temperature, the 30 degrees C-chilled birds having the highest temperature, and the 20 degrees C-chilled carcasses being intermediate but significantly different from either extreme. The carcass temperature differences at 105 min PM indicated that the carcass experienced differing chilling rates. To varying degrees, slower rates of chilling resulted in lower pH, greater degree of lightness (L* value), greater cook loss, and reduced gel strength. However, chilling rate had no effect on total protein solubility or myofibrillar phosphorylase for any of the treatments. Chilling rate seems to contribute to PSE turkey meat characteristics but by a mechanism independent of total protein solubility or myofibrillar phosphorylase.

Animals↗
PubMed1999

Protein Denaturation in Foam.

As part of a study of protein denaturation in foam we have measured the surface tension and the changes in protein structure occurring at the interface for lysozyme, pepsin, BSA, YADH, IgG, and catalase. The apparent CMC values were found to be dependent on the size and rigidity of the molecule. The variability of protein damage at a gas-liquid interface in foam was assessed using these proteins. The foams were produced under controlled conditions in a bubble column and were found to induce conformational changes in the protein molecules, but no fragmentation or disassociation of subunits occurred. Tertiary structural changes were detected in all the proteins studied, with some proteins forming aggregates. For pepsin, the secondary structure was also found to be altered. Enzyme solutions were used to determine the degree of biological activity retained after foaming for proteins with different structural characteristics. The more rigid proteins were found to display a low surface activity and a low degree of damage in foam. Pepsin suffered the highest rate of damage, which is thought to be a result of its inability to refold following denaturation. Copyright 1999 Academic Press.

Journal Article↗
PubMed1991

Immunoreactivity of cerebral amyloidosis is enhanced by protein denaturation treatments.

We investigated paraffin-embedded brain sections from three patients with Gerstmann-Sträussler syndrome and three patients with Alzheimer's disease or senile dementia of Alzheimer type using anti-human prion protein antisera and anti-beta/A4 protein antisera after protein denaturation treatments. After incubation with guanidine-thiocyanate, trichloroacetate, and phenol, the immunoreactivity of kuru plaques and senile plaques was enhanced to the same level as the formic acid treatment. These treatments revealed small compact amyloid deposits, amyloid deposits surrounding the plaque cores, and diffuse plaques. Most of these chemicals changed the congophilia of both amyloids. It is possible that these treatments denature amyloid fibril proteins and break down the structure of amyloid fibrils, thus revealing buried epitopes.

Adolescent↗
PubMed1996

A zinc finger-like domain of the molecular chaperone DnaJ is involved in binding to denatured protein substrates.

The Escherichia coli heat-shock protein DnaJ cooperates with the Hsp70 homolog DnaK in protein folding in vitro and in vivo. Little is known about the structural features of DnaJ that mediate its interaction with DnaK and unfolded polypeptide. DnaJ contains at least four blocks of sequence representing potential functional domains which have been conserved throughout evolution. In order to understand the role of each of these regions, we have analyzed DnaJ fragments in reactions corresponding to known functions of the intact protein. Both the N-terminal 70 amino acid 'J-domain' and a 35 amino acid glycine-phenylalanine region following it are required for interactions with DnaK. However, only complete DnaJ can cooperate with DnaK and a third protein, GrpE, in refolding denatured firefly luciferase. As demonstrated by atomic absorption and extended X-ray absorption fine structure spectroscopy (EXAFS), the 90 amino acid cysteine-rich region of DnaJ contains two Zn atoms tetrahedrally coordinated to four cysteine residues, resembling their arrangement in the C4 Zn binding domains of certain DNA binding proteins. Interestingly, binding experiments and cross-linking studies indicate that this Zn finger-like domain is required for the DnaJ molecular chaperone to specifically recognize and bind to proteins in their denatured state.

Absorptiometry, Photon↗
PubMed2005

The stress response against denatured proteins in the deletion of cytosolic chaperones SSA1/2 is different from heat-shock response in Saccharomyces cerevisiae.

BACKGROUND: A yeast strain lacking the two genes SSA1 and SSA2, which encode cytosolic molecular chaperones, acquires thermotolerance as well as the mild heat-shocked wild-type yeast strain. We investigated the genomic response at the level of mRNA expression to the deletion of SSA1/2 in comparison with the mild heat-shocked wild-type using cDNA microarray. RESULTS: Yeast cDNA microarray analysis revealed that genes involved in the stress response, including molecular chaperones, were up-regulated in a similar manner in both the ssa1/2 deletion mutant and the mild heat-shocked wild-type. Genes involved in protein synthesis were up-regulated in the ssa1/2 deletion mutant, but were markedly suppressed in the mild heat-shocked wild-type. The genes involved in ubiquitin-proteasome protein degradation were also up-regulated in the ssa1/2 deletion mutant, whereas the unfolded protein response (UPR) genes were highly expressed in the mild heat-shocked wild-type. RT-PCR confirmed that the genes regulating protein synthesis and cytosolic protein degradation were up-regulated in the ssa1/2 deletion mutant. At the translational level, more ubiquitinated proteins and proteasomes were detected in the ssa1/2 deletion mutant, than in the wild-type, confirming that ubiquitin-proteasome protein degradation was up-regulated by the deletion of SSA1/2. CONCLUSION: These results suggest that the mechanism for rescue of denatured proteins in the ssa1/2 deletion mutant is different from that in the mild heat-shocked wild-type: Activated protein synthesis in the ssa1/2 deletion mutant supplies a deficiency of proteins by their degradation, whereas mild heat-shock induces UPR.

Adenosine Triphosphatases↗
PubMed1988

Determination of molecular mass, Stokes' radius, frictional coefficient and isomer-type of non-denatured proteins by time-dependent pore gradient gel electrophoresis.

Molecular mass, Stoke's radius, frictional coefficient and isomer-type of non-denatured proteins can be obtained by time-dependent gradient gel electrophoresis by evaluating the resulting data using a two-step mathematical procedure. Provided a histochemical staining procedure is available to locate the position of an enzyme in the gel, crude cell extracts can be used for estimating their molecular size properties. The computation of molecular properties of non-denatured proteins is demonstrated for isozymes of aspartate aminotransferase (EC 2.6.1.1), peroxidase (EC 1.11.1.42) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from current-year needles of spruce. The resulting data as well as those which were calculated for esterase (EC 3.1.1.1), glutamate dehydrogenase (EC 1.4.1.4), isocitrate dehydrogenase (EC 1.4.1.42), and shikimate dehydrogenase (EC 1.1.1.25) are in accordance with those reported in the literature. The method described may be applied to various scientific areas such as genetics or environmental pollution. It could be shown here that current-year needles of injured spruce (damage class 3) contained two more peroxidase isozymes and one more glucose-6-phosphate dehydrogenase isozyme than those from non-injured trees. These differences may mark two genotypes of spruce of different susceptibilities towards present-day air and soil pollutants.

Calibration↗
PubMed2002

Protein denaturant binding polynomials.

We show how moments of the denaturant binding distribution function can be extracted from experimental data on the denaturation of a protein as a function of the concentration of denaturant and how in turn these moments can be used to construct the denaturant binding distribution function. This approach is similar to our recent work on using the maximum-entropy method to construct ligand-binding distributions from moments obtained from titration curves for nucleic acids and proteins. As an example we take literature data on the denaturation of ferro- and ferricytochrome c by guanidine hydrochloride and from it construct the denaturant binding polynomial and binding distribution function for the unfolded protein.

Cytochrome c Group↗
PubMed1997

The effect of electrical stimulation on the water-holding, capacity and protein denaturation of two bovine muscles.

The effect of low-voltage electrical stimulation on the water-holding capacity and protein denaturation of bovine longissimus thoracis and semimembranosus was studied in eight electrically stimulated (85 V, 14 Hz, 15 s; immediately after slaughter) and eight nonstimulated Friesian Holstein bull carcasses. At 24 h postmortem longissimus thoracis and semimembranosus were sampled for drip loss, thaw loss, filter paper wetness, myofibrillar water-holding capacity, protein content of drip, protein solubility, myofibrillar ATPase-activities, and bound phosphorylase. Electrical stimulation resulted in higher (P < .05) drip losses and filter paper wetness and lower (P < .05) myofibrillar water-holding capacity of semimembranous samples. The increased drip loss coincided with decreased (P < .05) sarcoplasmic protein solubility, decreased (P < .05) myofibrillar ATPase-activity, and decreased (P < .05) protein concentration of drip. In the longissimus thoracis, electrical stimulation resulted in a lower (P < .05) myofibrillar water-holding capacity. However, this was not reflected in higher drip losses. In this muscle, only a decreased (P < .05) sarcoplasmic protein solubility and increased (P < .05) amount of bound phosphorylase was observed. The results indicated that the negative effect of electrical stimulation on drip loss is possibly the result of myosin denaturation, which is determined by postmortem pH and temperature fall and thus varies by muscle studied. Sarcoplasmic protein denaturation seemed not to be involved in determining water-holding capacity.

Adenosine Triphosphatases↗
PubMed1976

Does Freund's adjuvant denature protein antigens? EPR studies of emulsified hemoglobin.

Use of complete Freund's adjuvant for production of antibodies to study protein conformation is valid only if emulsification in adjuvant does not denature protein antigens. Using electron paramagnetic resonance to observe directly the protein in situ in the opaque emulsions, we demonstrate that hemoglobin is not denatured by emulsification or storage in adjuvant for 24 hr at 4 degrees C, conditions comparable to the usual handling of antigens before immunization.

Antigens↗
PubMed2001

Non-polar solutes in water and in aqueous solutions of protein denaturants. Modeling of solution and transfer processes.

A simple molecular model for the thermodynamic behavior of non-polar solutes in water and in aqueous solutions of protein denaturants is presented. Three contributions are considered: (i) combinatorial arising from the mixing process, (ii) interactional characterizing the molecular interactions occurring in the mixture and (iii) a contribution originating from the structural changes occurring in the first shell of water molecules around the solute. The latter is modeled assuming that water molecules in contact with the solute are involved in a chemical equilibrium between two states. The model describes well the temperature and denaturant concentration dependences of the Gibbs energies of solution and transfer for benzene, toluene and alkanes in water and aqueous solutions of urea and guanidine hydrochloride. Model parameters are physically meaningful, allowing a discussion of the molecular interactions involved. A preferential solvation of the solute by the denaturant is found. However, the non-polar solute-denaturant interaction is not specific, i.e. leading to a distinct chemical entity. Urea and guanidine hydrochloride are non-polar solubilizing agents because their interactions with the solute are less unfavorable than those between water and the solute.

Hydrogen Bonding↗
PubMed1997

Heat shock protein induction by certain chemical stressors is correlated with their cytotoxicity, lipophilicity and protein-denaturing capacity.

Seven agents were analyzed with respect to their ability to induce heat shock protein (HSP) synthesis in C6 rat glioma cells. Induction of HSP synthesis was correlated with cytotoxicity and lipophilicity of the substances. In addition to the first four n-alcohols (methanol, ethanol, propanol and butanol) and phenol, whose capacity to induce HSP was analyzed earlier (Neuhaus-Steinmetz et al., 1994. Mol. Pharmacol. 45, 36-41), isopropanol, 1,4-dinitrophenol (DNP), diethylstilbestrol (DES), carbonylcyanide-m-chlorophenylhydrazone (CCCP), rotenone, paracetamol and acetyl salicylic acid (ASA) induced HSP synthesis after a 1-h incubation at a substance-specific concentration. The maximal induction of HSPs was closely correlated with the cytotoxicity of all substances and occurred when cell viability was reduced to 75 +/- 11% of the controls. Cytotoxicity and the ability to induce HSP were correlated with the lipophilicity of the alcohols, phenol, rotenone and paracetamol. Calculation of the hypothetical membrane concentrations of these compounds yielded a nearly equal value (0.54 +/- 0.13 M), indicating that interaction of substances with lipophilic cellular compounds, such as membranes or lipophilic core regions of proteins, is a critical step leading to HSP induction. This assumption is supported by a correlation between HSP induction and protein denaturation by the different alcohols (Herskovits et al., 1970. J. Biol. Chem. 245, 2588-2598). We assume that the amount of misfolded proteins induced by these lipophilic agents is responsible for the induction of HSP synthesis. ASA, DNP and CCCP induced HSP at lower concentrations than substances with a similar lipophilicity, which may be due to effects which add to the misfolding of proteins or to other signal pathways.

Actins↗
PubMed2000

Effect of protein denaturation on void fraction in foam separation column.

Foam fractionation is a cost-effective process that uses air to extract protein from a liquid (in this case "crude" dilute egg-albumin solution). This article deals with how the void fraction (fraction of air in the aerated solution) of foam is affected by heat denaturation of the protein. A 2-mm glass tube was used to sample the foam-liquid interface fluid in a 35-mm-diameter column in order to detect small changes in void fraction and foam production, which are not easily detected directly from the bulk foam. The main control variable in this study was the protein solution preheating time. As the preheating time increased, the initial void fraction in the column decreased. The initial void fraction of the undenatured solution ranged from about 0.73 to 0.80, and the void fraction for significant preheating times of 5 min ranged from approx 0.68 to 0.72. Furthermore, the period of foam production increased from 5 to 7 min for undenatured proteins in solution to as long 15 min for 5-min preheated solutions. Side-port sampling through a small capillary tube has the potential to be used as a rapid and inexpensive way to determine the level of protein denaturation by directly determining the void fraction and then estimating the effect of denaturation from a protein denaturation calibration curve of the void fraction.

Biotechnology↗
PubMed1997

Reactivation of denatured proteins by domain V of bacterial 23S rRNA.

In vitro transcripts containing domain V of the 23S rRNA of Escherichia coli and Bacillus subtilis can reactivate denatured proteins almost as efficiently as the total 23S rRNA. Here we show that almost the full length of domain V is required for reactivation of denatured pig muscle lactate dehydrogenase and pig heart cytoplasmic malate dehydrogenase: the central loop of this domain alone is not enough for this purpose. The antibiotic chloramphenicol, which binds to domain V of 23S rRNA, can inhibit reactivation of these proteins completely. Activity is eliminated by EDTA at a concentration of <1 mM, even in the presence of 4 mM MgCl2, suggesting that the three-dimensional conformation of the RNA should be maintained for this activity.

Animals↗
PubMed2001

Functional dissection of trigger factor and DnaK: interactions with nascent polypeptides and thermally denatured proteins.

In Escherichia coli, the ribosome-associated Trigger Factor (TF) cooperates with the DnaK system in the folding of newly synthesized cytosolic polypeptides. Here we investigated the functional relationship of TF and DnaK by comparing various functional properties of both chaperones. First, we analyzed the ability of TF and DnaK to associate with nascent polypeptides and full-length proteins released from the ribosome. Toward this end, we established an E. coli based transcription/translation system containing physiological ratios of TF, DnaK and ribosomes. In this system, TF can be crosslinked to nascent polypeptides of sigma32. No TF crosslink was found to full-length sigma32, which is known to be a DnaK substrate. In contrast, DnaK crosslinked to both nascent and full-length sigma32. DnaK crosslinks critically depended on the type of chemical crosslinker. Crosslinks represent specific substrate-chaperone interactions since they relied on the association of the nascent polypeptides with the substrate binding pocket of DnaK. While DnaK is known to be the major chaperone to prevent protein aggregation under heat shock conditions, we found that TF did not prevent aggregation of thermally unfolded proteins in vitro and was not able to complement the heat-sensitive phenotype of a deltadnaK52 mutant in vivo. These data indicate that TF and DnaK show strong differences in their ability to prevent aggregation of denatured proteins and to associate with native like substrates, but share the ability to associate with nascent polypeptides.

Bacterial Outer Membrane Proteins↗
PubMed1994

How random is a highly denatured protein?

There has been renewed interest in determining the physicochemical properties of denatured states of proteins. In many denatured states there is evidence for the existence of nonrandom configurational distributions. Here we examine the small-angle neutron scattering profile of yeast phosphoglycerate kinase in the native state and in highly denaturing conditions. We show that the denatured protein scattering profile can be interpreted using a model developed for synthetic polymers in which the chain behaves as a random coil in a good solvent, i.e. with excluded volume interactions. The implications of this result for our appreciation of the protein folding process are discussed.

Journal Article↗