Are proteins denatured by heat treatment?
Most proteins are denatured by heat treatment, and the process is usually irreversible. However, some proteins, such as hyperthermophilic proteins are known to be stable even at the boiling temperature of water. We here describe a systematic investigation of …
How much salt is needed to stabilize a protein?
Jan 18, 2012 · One of the most widely used thermodynamic models for pH effects on proteins was created by Tanford and coworkers, who showed that from knowledge of the pK a values of titratable groups it is possible to predict the change in protein native state stability as a function of pH 17. This thermodynamic model cannot be used to calculate the ...
How does pH affect protein-protein interactions in solution?
Jan 01, 2020 · The net effect of salts on protein stability is protein-dependent and is a complex balance of the multiple mechanisms by which the ionic salt interacts with protein molecules, shielding charged solvent exposed residues and then potentially decreasing protein-protein long-range electrostatic interactions.47, 81, 122 This charge shielding may be ...
What is the best thermodynamic model for the pH of proteins?
Apr 15, 2006 · Our findings suggest that the protein is most stable close to its isoelectric point (pI), and that 2 M NaCl does not eliminate the pH dependence of the stability; and also suggest that even at high salt, the pK a values may differ between folded and unfolded proteins. Materials and methods Cloning and expression of PGB1-QDD
What has the greatest influence on protein stability?
Temperature and pH have the greatest influence on both physical and chemical protein stability. High temperatures can lead to thermal denaturation and subsequent aggregation as well as accelerate chemical degradation pathways (i.e., side chain oxidation, hydrolysis, and deamidation) that may result in aggregation.Aug 14, 2015
What factors affect protein stability?
Many factors affect the process of protein folding, including conformational and compositional stability, cellular environment including temperature and pH, primary and secondary structure, solvation, hydrogen bonding, salt bridges, hydrophobic effects, van der Waals (vdW) forces, ligand binding, cofactor binding, ion ...Jan 26, 2016
How does pH affect protein stability?
Hydrogen Bonding Changing the pH disrupts the hydrogen bonds, and this changes the shape of the protein.Jul 1, 2014
How does salt affect protein stability?
The presence of high salt in a protein solution will have the following implications: disturbance in local water structure around the protein; decreased propensity for intermolecular hydrogen bonds, affecting protein solubility, binding, stability and crystallization; increased surface tension of water, striping off ...Apr 9, 2014
What are the two main factors that stabilize the proteins in solutions?
The literature is in general agreement that the two types of interaction that are most prevalent in proteins are (i) hydrophobic interactions and (ii) hydrogen bonds.
How can you increase the stability of a protein?
It is widely accepted that the solubility and stability of proteins can be increased by the use of additives in buffers (e.g., ionic compounds, salts, detergents, osmolytes, etc).Jul 1, 2004
What pH are proteins most stable?
A typical example is extracting the pH of maximal stability of 8.0 from a paper that states that the “protein was stable up to pH 8.0”.
How does salt concentration affect protein structure?
High salt concentrations promote the aggregation and precipitation of proteins. This phenomenon is considered to occur as a result of disruption of the hydration barriers between protein molecules, as salt causes water surrounding the protein to move into the bulk solution.
How does pH denature a protein?
Posted June 22, 2020. Acids and bases can significantly change the environmental pH of proteins, which disrupts the salt bridges and hydrogen bonding formed between the side chains, leading to denaturation.Jun 22, 2020
How does salt concentration affect protein solubility?
Salt Effects Solubility at low ionic strength increases with salt concentration. Pairing of salt ions with charged groups of the protein shields intramolecular repulsion. Precipitates tend to be irreversible, suggesting that the protein is denatured. Solubility at high ionic strength decreases with salt concentration.Aug 23, 1999
Does salt denature protein?
In summary, depending on the salt and the concentration, salt can denature a protein by competing for electrostatic interactions within the protein replacing them with protein-salt interactions or disrupt the structure of water that allows both the grease and charge to weaken.Dec 3, 2021
Does salting denature proteins?
The starting molecules strengthen hydrophobic interactions by decreasing solubility of the nonpolar molecules, thus salting out the system. However, the later molecules begin to denature the structure of the protein because of strong ionic interactions that disrupt hydrogen bonding.
What temperature should a protein be stored?
This temperature is in most cases between 40°C and 80°C and manufacturers generally recommend storing biopharmaceuticals between 2°C and 8°C, well below this Tm. 29.
What is protein denaturation?
Protein denaturation refers to the loss of higher-order structure through unfolding. It may result from previously described chemical instabilities, or from environmental conditions such as extreme temperatures or pH. The consequences of unfolding can be direct perturbation of the mAb’s function, for example, a decrease in hinge flexibility, or promotion of aggregation. 28
What is monoclonal antibody?
Monoclonal antibodies are immunoglobulins (or fragments of immunoglobulins) with a precise target, produced from a single cellular clone. 2 They are proteins composed of 4 chains—2 light chains and 2 heavy chains—linked together with disulfide bridges.
What is disulfide bond?
Oxidation, including disulfide bond formation, is one of the most frequent chemical degradation. It may happen in presence of oxidants (such as peroxides, light, or metals) or without, then referred to as auto-oxidation. 28 Some residues are particularly susceptible to oxidation, including methionine, histidine, and cysteine residues. 28, 31 Disulfide bond formation is one of the consequences of cysteine oxidation that occurs between 2 oxidized free residues, with a thiolate anion intermediate. 24 The formation of those bridges may be intramolecular or intermolecular, and is enhanced in basic environment. 28
How does protein concentration affect aggregation?
Higher protein concentrations also seem to increase the viscosity of solutions, which itself may increase the aggregation potential of proteins by enhancing protein-protein interactions and self-association. 27, 62 This concentration-dependent tendency to aggregation is an increasing concern considering the extended use of subcutaneous administration of mAbs which require highly concentrated solutions. However, the real impact of high protein concentrations is complex, as can be seen, for example, in the work published by Hauptmann et al. 63 where they showed that high concentrations increased smaller particles concentrations while decreasing bigger ones, whereas Nicoud et al. 64 showed an increase in aggregation rate with concentration. However, other studies have reported more mitigated results, for example, where increasing protein concentration decreased the rate of aggregation. 24
How are proteins exposed to light?
Protein medications are exposed to light at many points during their life. For example, during production they are generally purified by column chromatography and then are exposed (briefly) to the ultraviolet (UV) light of the detector, but the main exposure to light occurs during storage and during administration to patients with IV-bags. The exposition may be even more important if the primary container is not stored within an opaque secondary container. Proteins, especially their aromatic residues, are very sensible to light, inducing photodegradation, mainly through photo-oxidation and formation of oxygenated radicals, but also fragmentation and cross-linking. 106 Antibodies, containing a large number of aromatic residues (especially tryptophan residues), are particularly sensible to this phenomenon, confirming the greater impact of UV light when compared to white light. On the other hand, light exposure does not seem to directly alter mAbs secondary and tertiary structures. 107 Liquid forms also seem to be more sensible than lyophilized forms. 108
What is the importance of amino acid sequence?
The amino acid sequence (primary structure) gives an important clue on whether a protein will be prone or not to aggregation. For example, a low isoelectric point (pI) of CDR seems to promote soluble aggregates formation by enhancing electrostatic interactions between mAbs, while a high pI of CDR leads preferentially to insoluble aggregates, especially if in contact with negatively charged surfaces. 57
What is the pI of PGB1-QDD?
The pI of PGB1-QDD was determined by isoelectric focusing at low salt concentration and found to be 4.3 ± 0.1 (data not shown). The pI, based on model pK a values for individual amino acids (31), is 4.2. This corresponds to a protein where all electrostatic interactions are shielded. Hence, there is no difference in pI between the model and experiment. Wt PGB1 is reported to have a pI of 4.0 determined by isoelectric focusing (25). In this case, the calculated pI is 4.4 based on model pK a values and 4.2 based on measured pK a values (32). We find that pI is, within error, the same for wt and PGB1-QDD despite the introduction of two negative charges.
Does salt affect protein stability?
Protein surface charges are modulated by pH variations, but the addition of salt may attenuate the effects on protein stability. We have measured protein stability as a function of pH at low, physiological, and high salt concentrations. PGB1 is an excellent model protein.
Abstract
Reconstituted milk protein concentrate (RMPC) showed higher thermal stability than reconstituted skimmed milk powder (RSMP) at equal protein level (7.5%); heat coagulation time (HCT) 6.80 and 1.80 min, respectively. RMPC contained lower concentration of milk minerals as compared to RSMP.
1. Introduction
Some milk products such as fluid milk and milk protein beverages are thermally processed before packaging. These dairy product formulation requires to have an adequate thermal processing stability.
2. Materials and methods
Commercial low heat SMP and MPC powders were used to prepare all milk protein dispersions. These powders were supplied by Maxum Foods Pty. Ltd, Australia. All reagents were procured from Sigma-Aldrich Pty. Ltd., Sydney, NSW, Australia, unless stated otherwise. Recipe for simulated milk ultrafiltrate (SMUF) was adopted from Jenness (1962).
4. Conclusions
The results from this work showed that addition of mineral salts such as NaCl, KCl, CaCl 2 and MgCl 2 in RMPC affected its UHT stability.
Credit author statement
Jaspal Singh: Conceptualization, Methodology, Investigation, Formal analysis, Visualization, Writing – original draft. Agathe Dean: Conceptualization, Methodology, Investigation, Formal analysis, Visualization, Writing – original draft. Sangeeta Prakash: Conceptualization, Methodology, Supervision.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
The authors would like to acknowledge the support provided by “University of Queensland Centennial Scholarship” and “Australian Government Research Training Program Scholarship”.
What is the stability of PTEN?
The protein stability of PTEN in the cell is a factor in its signaling properties and PIP3 control. When PTEN C-terminal phosphorylation was discovered, it was also found that there was a correlation between its phosphorylation state and protein stability (Vazquez et al., 2000). This behavior is somewhat paradoxical as the enzymatic activity of phospho-PTEN is impaired which means that the more stable PTEN form is also less functional in PIP3 hydrolysis. Furthermore, the molecular basis of PTEN protein destruction was not yet clear in this early work. Cellular protein clearance often involves Lys ubiquitination which then targets the protein for proteasome-mediated degradation. Ubiquitination is a three-step process involving E1, E2, and E3 ubiquitin transferring enzymes. Several ubiquitin E3 ligases for PTEN have been reported, and the two most extensively studied have been NEDD4-1 (Wang et al., 2007) and WWP2 ( Maddika et al., 2011). NEDD4-1 and WWP2 are both HECT domain E3 ligases and are composed of an N-terminal C2 domain followed by four WW domains and culminating in catalytic HECT domains.
How is protein stability modulated?
Protein stability is commonly modulated through variations in solution conditions, a strategy that has relevance in applications to protein overexpression on a small scale, in applications to ligand binding and in applications to commercial formulation in the development of therapeutic proteins .
Why are membrane proteins unstable?
Many membrane proteins are unstable in detergent solution. The stability often depends critically on the choice of detergent and on the presence of lipids. Strict requirements for a particular detergent or lipid may make 3D crystallization difficult, because either may interfere with crystal contacts. These requirements are easily accommodated in ...
What is the most important factor in the development of transgenic plants?
Protein stability is an often-overlooked component of transgene expression, but in many cases this is the most crucial factor in the development of useful transgenic plants. Proteins emerging from the ribosome must fold to achieve their native conformation, they are often chemically modified, and some must then assemble into multiprotein complexes. These posttranslational processes are interdependent and often depend critically on the site within the cell where the protein accumulates. Therefore a failure of any one process can lead to the accumulation of a misfolded and nonfunctional polypeptide that is rapidly degraded.