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How to denature a protein

Protein denaturation in reverse micellar systems is an important aspect that needs to be carefully considered.

Related terms:

  • Peptide
  • Heat Shock Proteins
  • Nested Gene
  • pH
  • Denaturation
  • Solubility
  • Digestion

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Denatured corona proteins mediate the intracellular bioactivities of nanoparticles via the unfolded protein response

Shang Liu , . Lei Dong , in Biomaterials , 2020

Abstract

Biomolecular corona formed on nanoparticles (NPs) influences the latter’s in vivo biological effects. Nanomaterials with different physicochemical properties exert similar adverse effects, such as cytotoxicity, suggesting the existence of ubiquitous signals during various corona formations that mediate common and fundamental cellular events. Here, we discover the involvement of the unfolded protein response (UPR) and recruited chaperones in the corona. Specially, heat shock protein 90 kDa α class B member 1 (Hsp90ab1) is abundantly enriched in the corona, accompanied by substantial aggregation of misfolded protein on particles intracellularly. Further analysis reveals the particulate matter 2.5 (PM2.5) and metal-containing particles are more capable of denaturing proteins. The recruited Hsp90ab1 activates diverse NPs’ pathological behaviour by heat stress response (HSR), which were significantly reversed by geldanamycin (GA), the inhibitor of Hsp90ab1. Murine lung inflammation induced by PM2.5 and iron oxide NPs (Fe 3O 4NPs) is suppressed by GA, highlighting that Hsp90ab1-mediated UPR is a potential target for the treatment of environmental pollution-related illnesses. Based on our findings, the UPR and Hsp90ab1 presented in the corona of particles initiate fundamental intracellular reactions that lead to common pathological outcomes, which may provide new insights for understanding nanotoxicity and designing therapeutic approaches for diseases associated with environmental pollution.

Proteins, Proteomics, and the Dysproteinemias

D Handling and Identification of Proteins

Protein denaturation is the net effect of alterations in the biological, chemical, and physical properties of the protein by mild disruption of its structure. When blood samples are taken for protein analysis, it is important that they are handled correctly so that no artifacts are introduced that could affect the investigation and its interpretation. If the protein is allowed to even partially degrade, the assay will not be accurate. Therefore, it is essential that denaturation is avoided. The ability of plasma proteins to resist denaturation in a blood sample taken for diagnostic analysis varies between proteins; consequently, the sample should be handled according to the analysis required. Fortunately, most major plasma proteins are relatively resistant to denaturation and can be assayed in samples that have been handled carefully and have been kept away from elevated temperatures. However, separation of plasma or serum from the blood cells by centrifugation should be performed as early as possible. Thereafter, many proteins are stable at 4°C for several days and at –20°C for much longer (months to years). Some proteins are less stable, with enzymes being particularly susceptible to loss of activity with time, while the stability of the peptide hormone ACTH is so low that samples should be snap-frozen immediately to preserve the intact peptide.

For identification and quantification of serum protein, the protein component in serum must either be separated or individual proteins must be measured independently. The primary separation of the proteins in serum is between albumin and the globulins. Albumin is a water-soluble, globular protein that is usually identifiable as a single discrete molecule. The globulins are also globular proteins, but many of them, in contrast to albumin, precipitate in pure water and require salts to maintain their solubility. The globulins are a mix of proteins of various types, which migrate in groups in an electric field (electrophoresis) as families of proteins identified as α-, β-, or γ-globulins. The nomenclature of the globulin fractions is based on their location during separation by electrophoresis. Albumin has the most rapid migration of the major proteins (in some species it is preceded by prealbumin), followed by the α-globulin, β-globulin, and γ-globulin fractions, respectively. The γ-globulins are largely composed of immunoglobulins, the antibodies that bind to invading pathogens or other foreign matter. In contrast, the α- and β-globulin fractions contain a great variety of different proteins.

Electrophoresis is a well-established diagnostic method that was first introduced to the clinical biochemistry laboratory with cellulose acetate as the support medium for the separation. This has largely been replaced with agarose, so that serum protein electrophoresis (SPE) in agarose gels, followed by protein staining and densitometry to quantify the protein in each of the main fractions, is common in clinical biochemistry laboratories. This has evolved into an extremely useful technique because aberrations are observed in many disease states though there are only a few diseases where the electrophoretic pattern can provide a definitive diagnosis.

Interest has advanced the investigation armory for serum protein analysis with the development of specific analytical methods for individual proteins. Though specific assays have been used for a long time for determination of proteins such as albumin and fibrinogen, it is only relatively recently that specific assays for other diagnostically useful proteins such as haptoglobin, CRP, SAA, and α1-acid glycoprotein (AGP) have become commonly available. In most cases, this has been achieved by the use of immunoassays, which has often required the development and validation of species-specific methodology.

When a solution of a protein is boiled, the protein frequently becomes insoluble—i.e., it is denatured—and remains insoluble even when the solution is cooled. The denaturation of the proteins of egg white by heat—as when boiling an egg—is an example of irreversible denaturation. The denatured protein has the same primary structure as the original, or native, protein. The weak forces between charged groups and the weaker forces of mutual attraction of nonpolar groups are disrupted at elevated temperatures, however; as a result, the tertiary structure of the protein is lost. In some instances the original structure of the protein can be regenerated; the process is called renaturation.

Denaturation can be brought about in various ways. Proteins are denatured by treatment with alkaline or acid, oxidizing or reducing agents, and certain organic solvents. Interesting among denaturing agents are those that affect the secondary and tertiary structure without affecting the primary structure. The agents most frequently used for this purpose are urea and guanidinium chloride. These molecules, because of their high affinity for peptide bonds, break the hydrogen bonds and the salt bridges between positive and negative side chains, thereby abolishing the tertiary structure of the peptide chain. When denaturing agents are removed from a protein solution, the native protein re-forms in many cases. Denaturation can also be accomplished by reduction of the disulfide bonds of cystine—i.e., conversion of the disulfide bond (―S―S―) to two sulfhydryl groups (―SH). This, of course, results in the formation of two cysteines. Reoxidation of the cysteines by exposure to air sometimes regenerates the native protein. In other cases, however, the wrong cysteines become bound to each other, resulting in a different protein. Finally, denaturation can also be accomplished by exposing proteins to organic solvents such as ethanol or acetone. It is believed that the organic solvents interfere with the mutual attraction of nonpolar groups.

Some of the smaller proteins, however, are extremely stable, even against heat; for example, solutions of ribonuclease can be exposed for short periods of time to temperatures of 90 °C (194 °F) without undergoing significant denaturation. Denaturation does not involve identical changes in protein molecules. A common property of denatured proteins, however, is the loss of biological activity—e.g., the ability to act as enzymes or hormones.

Although denaturation had long been considered an all-or-none reaction, it is now thought that many intermediary states exist between native and denatured protein. In some instances, however, the breaking of a key bond could be followed by the complete breakdown of the conformation of the native protein.

Although many native proteins are resistant to the action of the enzyme trypsin, which breaks down proteins during digestion, they are hydrolyzed by the same enzyme after denaturation. The peptide bonds that can be split by trypsin are inaccessible in the native proteins but become accessible during denaturation. Similarly, denatured proteins give more intense colour reactions for tyrosine, histidine, and arginine than do the same proteins in the native state. The increased accessibility of reactive groups of denatured proteins is attributed to an unfolding of the peptide chains.

If denaturation can be brought about easily and if renaturation is difficult, how is the native conformation of globular proteins maintained in living organisms, in which they are produced stepwise, by incorporation of one amino acid at a time? Experiments on the biosynthesis of proteins from amino acids containing radioactive carbon or heavy hydrogen reveal that the protein molecule grows stepwise from the N terminus to the C terminus; in each step a single amino acid residue is incorporated. As soon as the growing peptide chain contains six or seven amino acid residues, the side chains interact with each other and thus cause deviations from the straight or β-chain configuration. Depending on the nature of the side chains, this may result in the formation of an α-helix or of loops closed by hydrogen bonds or disulfide bridges. The final conformation is probably frozen when the peptide chain attains a length of 50 or more amino acid residues.

I know of Anfinsen’s experiments and I’m aware that some denatured enzymes may regain their lost activity through the removal of the denaturant agent. What I’m unaware of is how rare is it for a protein to be able to renature? Can all proteins do that? If not all of them can do it then how rare or common is it to find a protein which can be renatured?

P.S. To avoid answers like “depend on your denaturation process” let us assume that a protein can be renatured if it has been shown to renature in at least one experimental setup.

How to denature a protein

1 Answer 1

The answer is more like “It depends on the protein, and the renaturation (or refolding) process.” There are a lot of factors that contribute to an individual protein’s ability to refold, including size, sequence, secondary structure, amount and type of inter-amino acid links like disulfide bonds, number of subunits, the presence of chaperones/heat shock proteins, and, yes, how it was denatured in the first place (sorry, I couldn’t resist). Smaller proteins will refold more easily than larger ones. Hydrophilic proteins tend to refold better than more hydrophobic ones, especially membrane-bound proteins. Multi-subunit proteins/complexes tend to need some help to properly reassemble. Adding in chaperones/heat shock proteins will almost certainly help the process along for all but the smallest and hardiest samples, and will give you better results than just dialyzing all the salts/detergents/chaotropic agents away into PBS. Finally, if you denature by boiling in Laemmli buffer, you’re going to have a very difficult time refolding most things, while going from Guanidine HCl to PBS isn’t always that bad, depending on what you’re looking at.

So, unfortunately the answer is “it depends.” You have to remember that the intracellular environment is very complex, and is designed to correctly fold proteins after they’re made, and break down misfolded proteins before they can aggregate or cause other damage. There are hundreds of thousands of proteins just in humans, so it is rather difficult to make blanket statements, but for the sake of resistance to damage, many smaller, single-subunit proteins can likely be at least partly to mostly refolded and regain some or all of their original activity. As complexity grows, however, the likelihood of successfully regaining function decreases.

How to denature a protein

10kDa) are readily renatured and used in protein folding kinetics experiments. Its pretty unlikely that anything larger will be useful. Do you have any specific examples of a larger protein that has been refolded by any means, and if so do you think this is a typical result? $\endgroup$

Protein molecules deform and unravel when exposed to acid.

What are proteins?

Proteins are large molecules found in our bodies and food, consisting of many smaller components called amino acids. Proteins have the properties they do because of the shape and arrangement of their amino acids. A weak bond, known as a hydrogen bond, forms between a hydrogen atom and an oxygen atom in the amino acids. This gives the protein its shape.

What is denaturing and how does it happen?
A protein becomes denatured when its normal shape gets deformed because some of the hydrogen bonds are broken. Weak hydrogen bonds break when too much heat is applied or when they are exposed to an acid (like citric acid from lemon juice). As proteins deform or unravel parts of structure that were hidden away get exposed and form bonds with other protein molecules, so they coagulate (stick together) and become insoluble in water. Curing salmon using lemon and lime juice (eg. to make a gravadlax or ceviche) is an example of protein acid denaturation.

  1. Place an egg white into a clean bowl
  2. Observe the colour and texture of the egg white
  3. Now add 3ml of lemon juice to the egg white and stir
  4. Record what happens to the colour and texture of the egg white

Egg white turns solid and goes white instead of clear when it denatures

More information

Meat and Education.com worksheet: What makes meat tender

Meat and Education.com teachers’ notes: What happens when meat is cooked

OCR topic exploration pack: Preparation techniques

How to denature a protein

You’ve nurtured your cells for weeks, perfected your experimental conditions, and nailed down all the controls. You’ve harvested your cells and gently lysed them, now you’re ready to look at the proteins. What’s one of the most common next step in protein analysis? A denaturing gel or SDS-Polyacrylamide Gel Electrophoresis!

SDS-Polyacrylamide Gel Electrophoresis, or SDS-PAGE for short, is the technique where proteins are denatured and linearized, then run across a current through a thin gel, which separates the proteins by size. SDS-PAGE is a key step in many experiments including:

  • Western Blots
  • Separating proteins for mass spectrometry
  • Determining protein purity (such as after an immunoprecipitation or protein purification)
  • Examining protein size
  • Quantifying protein abundance
  • Visualizing post-translational modifications

…. Basically just about any experiment where you need to know something about your favorite proteins. SDS-PAGE is an essential lab tool you’ll want to master. And part of mastering it is understanding how it works.

Unraveling the Proteins

The first step in running a denaturing gel is to denature your proteins. This is accomplished using:

When you have your proteins in hand — whether they are from a cell lysate or purified sample — denaturing your proteins is the first step and for this you need Sodium dodecyl sulfate (SDS). SDS is the main star of the denaturing protein gel. SDS is a detergent composed of a hydrophobic hydrocarbon tail attached to an ionic sulphate group and a key component of loading buffer,. When SDS meets up with your protein, SDS’s hydrocarbon tail dissolves any hydrophobic region of the protein, while the sulfate end breaks non-covalent ionic bonds. This causes your protein to lose its secondary and tertiary structure, and well…unfold. Once surrounded by SDS, your previously carefully folded protein becomes loose and long, just like overcooked spaghetti.

A Reducing Agent

Also in the denaturing mix is the noxious ?-mercaptoethanol (?-me). Its job is to break disulphide bonds, which further spaghettifies your protein. Its strong smell also lets everyone else in the lab know you’re doing a protein gel!

The final factor in denaturing your protein is heat. Typically, you will boil your protein samples in the loading buffer (containing Tris-HCl, SDS, bromophenol blue, glycerol, and ?-me) before loading them in your gel. This helps to completely denature the proteins and also helps with physically loading the gel. Protein samples frequently are gummy, particularly if the protein prep is from cell or tissue extracts and therefore contains DNA. Boiling homogenates your sample, as the heat melts any DNA in the prep, in turn making it less gummy and easier to load on the gel.

Separating the Proteins

Now that your proteins are nice and linear, it’s time to run them out on your polyacrylamide gel using electricity. That’s right it is time for PolyAcrylamide Gel Electrophoresis or PAGE.

The Resolving Gel

The main component of PAGE is the resolving acrylamide gel. Acrylamide gels are composed of SDS, buffer, ammonium persulfate, and TEMED. This mixture is poured between two closely spaced plates, forming a thin gel. Once polymerized this mix creates a sieve-like network that the proteins can then travel through. As the proteins run through the gel, they will work through the mesh structure of the gel. Larger proteins take longer to navigate through the gel, while smaller proteins move faster. Therefore, you can adjust how much proteins will separate by changing the percentage of acrylamide in the gel. Less acrylamide means a gel with larger pores, which good for large proteins. More acrylamide means a gel with smaller pores, which is great for separating smaller proteins.

The Current

The proteins don’t move through the gel by their own volition. Instead you must subject your gel to an electric current, with the negative charge at the top where the proteins are loaded and the positive charge at the bottom. SDS-coated proteins have a large negative charge (thanks to the SDS), thus the proteins are attracted to the positive charge and move from top to bottom.

The Stacking Gel

A stacking gel is poured at the top of the gel. The stacking gel is made of the same stuff as the resolving gel but with a lower concentration of acrylamide. It is poured, after your resolving gel polymerizes, immediately before loading the gel. This is where you place your loading comb, which will create neat wells for your protein sample. A decent stacking gel is important to ensure crisp, sharp bands on your gel. The stacking gel ensures that, regardless of protein size and sample volume, all your proteins enter the gel at the same time.

A Few Cautions

Heat is a friend and foe.

There are two points during an SDS-PAGE where you need to keep an eye on the temperature. First, is boiling your sample. Larger proteins may need a longer boiling time to facilitate denaturing, while smaller proteins may degrade with too much heat. Therefore, it’s a good idea to test your boiling time when using a new protein sample. The second heat issue is during the gel run. Running a current across the gel generates heat. Running the gel too fast, at too high of a current, can over-heat your gel causing it to warp or even melt. This will not make for a pretty gel, so try to avoid cranking up the current because you want to go to lunch.

Birds of a Feather Flock Together

Proteins of nearly the same size will migrate at the same rate. If you are looking for two similarly sized proteins, or trying to visualize small post-translational modifications, you may need to tweak the acrylamide percentage up for better resolution, or run your sample longer.

And there you have SDS-PAGE! When your gel is finished running, carefully separate the plates and move on to the next step of your experiment!

Has this helped you? Then please share with your network.

The onset of protein denaturation and loss of cell structure is time dependent and begins to occur above 318.15K (Bilchik et al., 2001), which correlates to an increase in temperature of 8K above the physiological value.

Related terms:

  • Monospecific Antibody
  • pH
  • Denaturation
  • Fluorescence
  • Ultrasound
  • Mutation
  • Microorganism
  • Cell Membrane

Download as PDF

About this page

Denatured corona proteins mediate the intracellular bioactivities of nanoparticles via the unfolded protein response

Shang Liu , . Lei Dong , in Biomaterials , 2020

Abstract

Biomolecular corona formed on nanoparticles (NPs) influences the latter’s in vivo biological effects. Nanomaterials with different physicochemical properties exert similar adverse effects, such as cytotoxicity, suggesting the existence of ubiquitous signals during various corona formations that mediate common and fundamental cellular events. Here, we discover the involvement of the unfolded protein response (UPR) and recruited chaperones in the corona. Specially, heat shock protein 90 kDa α class B member 1 (Hsp90ab1) is abundantly enriched in the corona, accompanied by substantial aggregation of misfolded protein on particles intracellularly. Further analysis reveals the particulate matter 2.5 (PM2.5) and metal-containing particles are more capable of denaturing proteins. The recruited Hsp90ab1 activates diverse NPs’ pathological behaviour by heat stress response (HSR), which were significantly reversed by geldanamycin (GA), the inhibitor of Hsp90ab1. Murine lung inflammation induced by PM2.5 and iron oxide NPs (Fe 3O 4NPs) is suppressed by GA, highlighting that Hsp90ab1-mediated UPR is a potential target for the treatment of environmental pollution-related illnesses. Based on our findings, the UPR and Hsp90ab1 presented in the corona of particles initiate fundamental intracellular reactions that lead to common pathological outcomes, which may provide new insights for understanding nanotoxicity and designing therapeutic approaches for diseases associated with environmental pollution.

Proteins, Proteomics, and the Dysproteinemias

D Handling and Identification of Proteins

Protein denaturation is the net effect of alterations in the biological, chemical, and physical properties of the protein by mild disruption of its structure. When blood samples are taken for protein analysis, it is important that they are handled correctly so that no artifacts are introduced that could affect the investigation and its interpretation. If the protein is allowed to even partially degrade, the assay will not be accurate. Therefore, it is essential that denaturation is avoided. The ability of plasma proteins to resist denaturation in a blood sample taken for diagnostic analysis varies between proteins; consequently, the sample should be handled according to the analysis required. Fortunately, most major plasma proteins are relatively resistant to denaturation and can be assayed in samples that have been handled carefully and have been kept away from elevated temperatures. However, separation of plasma or serum from the blood cells by centrifugation should be performed as early as possible. Thereafter, many proteins are stable at 4°C for several days and at –20°C for much longer (months to years). Some proteins are less stable, with enzymes being particularly susceptible to loss of activity with time, while the stability of the peptide hormone ACTH is so low that samples should be snap-frozen immediately to preserve the intact peptide.

For identification and quantification of serum protein, the protein component in serum must either be separated or individual proteins must be measured independently. The primary separation of the proteins in serum is between albumin and the globulins. Albumin is a water-soluble, globular protein that is usually identifiable as a single discrete molecule. The globulins are also globular proteins, but many of them, in contrast to albumin, precipitate in pure water and require salts to maintain their solubility. The globulins are a mix of proteins of various types, which migrate in groups in an electric field (electrophoresis) as families of proteins identified as α-, β-, or γ-globulins. The nomenclature of the globulin fractions is based on their location during separation by electrophoresis. Albumin has the most rapid migration of the major proteins (in some species it is preceded by prealbumin), followed by the α-globulin, β-globulin, and γ-globulin fractions, respectively. The γ-globulins are largely composed of immunoglobulins, the antibodies that bind to invading pathogens or other foreign matter. In contrast, the α- and β-globulin fractions contain a great variety of different proteins.

Electrophoresis is a well-established diagnostic method that was first introduced to the clinical biochemistry laboratory with cellulose acetate as the support medium for the separation. This has largely been replaced with agarose, so that serum protein electrophoresis (SPE) in agarose gels, followed by protein staining and densitometry to quantify the protein in each of the main fractions, is common in clinical biochemistry laboratories. This has evolved into an extremely useful technique because aberrations are observed in many disease states though there are only a few diseases where the electrophoretic pattern can provide a definitive diagnosis.

Interest has advanced the investigation armory for serum protein analysis with the development of specific analytical methods for individual proteins. Though specific assays have been used for a long time for determination of proteins such as albumin and fibrinogen, it is only relatively recently that specific assays for other diagnostically useful proteins such as haptoglobin, CRP, SAA, and α1-acid glycoprotein (AGP) have become commonly available. In most cases, this has been achieved by the use of immunoassays, which has often required the development and validation of species-specific methodology.

How to denature a protein

Protein molecules carry out many important tasks in living systems. Most important, the majority of proteins are quite specific about which task they perform. Protein structure is what dictates this specificity, and the three-dimensional (tertiary) structure is particularly important. When this specific three-dimensional structure is disrupted, the protein loses its functionality and is said to have undergone denaturation.

The interactions, such as hydrogen bonding , that dictate the tertiary structure of proteins are not as strong as covalent chemical bonds. Because these interactions are rather weak, they can be disrupted with relatively modest stresses.

If a solution containing a protein is heated, it will reach a temperature at which properties such as viscosity or the absorption of ultraviolet (UV) light will change abruptly. This temperature is called the melting temperature of the protein (because the measurement is analogous to that made for the melting of a solid). The melting temperature varies for different proteins, but temperatures above 41°C (105.8°F) will break the interactions in many proteins and denature them. This temperature is not that much higher than normal body temperature (37°C or 98.6°F), so this fact demonstrates how dangerous a high fever can be.

A familiar example of heat-caused denaturation are the changes observed in the albumin protein of egg whites when they are cooked. When an egg is first cracked open, the “whites” are translucent and runny (they flow like a liquid), but upon heating they harden and turn white. The change in viscosity and color is an indication that the proteins have been denatured.

Factors other than heat can also denature proteins. Changes in pH affect the chemistry of amino acid residues and can lead to denaturation. Hydrogen bonding often involves these side changes. Protonation of the amino acid residues (when an acidic proton H + attaches to a lone pair of electrons on a nitrogen) changes whether or not they participate in hydrogen bonding, so a change in the pH can denature a protein.

How to denature a protein

How to denature a protein

Changes in salt concentration may also denature proteins, but these effects depend on several factors including the identity of the salt. Some salts, such as ammonium sulfate, tend to stabilize protein structures and increase the melting temperature. Others, such as calcium chloride, destabilize proteins and lower the melting temperature and are called chaotropic. Salts in this category can also be used in the laboratory to help purify proteins that are being studied, by lowering their solubility and causing them to “salt out.”

Thomas A. Holme

Bibliography

Branden, C., and Tooze, J. (1998). Introduction to Protein Structures, 2nd edition. London: Taylor and Francis.

Creighton, T. E. (1993). Proteins, 2nd edition. New York: Freeman.

Fagain, C. O. (1997). Protein Stability and Stabilization of Protein Function. Georgetown, TX: Landes Bioscience.

Protein molecules carry out many important tasks in living systems. Most important, the majority of proteins are quite specific about which task they perform. Protein structure is what dictates this specificity, and the three-dimensional (tertiary) structure is particularly important. When this specific three-dimensional structure is disrupted, the protein loses its functionality and is said to have undergone denaturation.

The interactions, such as hydrogen bonding , that dictate the tertiary structure of proteins are not as strong as covalent chemical bonds. Because these interactions are rather weak, they can be disrupted with relatively modest stresses.

If a solution containing a protein is heated, it will reach a temperature at which properties such as viscosity or the absorption of ultraviolet (UV) light will change abruptly. This temperature is called the melting temperature of the protein (because the measurement is analogous to that made for the melting of a solid). The melting temperature varies for different proteins, but temperatures above 41 ° C (105.8 ° F) will break the interactions in many proteins and denature them. This temperature is not that much higher than normal body temperature (37 ° C or 98.6 ° F), so this fact demonstrates how dangerous a high fever can be.

A familiar example of heat-caused denaturation are the changes observed in the albumin protein of egg whites when they are cooked. When an egg is first cracked open, the “whites” are translucent and runny (they flow like a liquid), but upon heating they harden and turn white. The change in viscosity and color is an indication that the proteins have been denatured.

Factors other than heat can also denature proteins. Changes in pH affect the chemistry of amino acid residues and can lead to denaturation. Hydrogen bonding often involves these side changes. Protonation of the amino acid residues (when an acidic proton H + attaches to a lone pair of electrons on a nitrogen) changes whether or not they participate in hydrogen bonding, so a change in the pH can denature a protein.

Changes in salt concentration may also denature proteins, but these effects depend on several factors including the identity of the salt. Some salts, such as ammonium sulfate, tend to stabilize protein structures and increase the melting temperature. Others, such as calcium chloride, destabilize proteins and lower the melting temperature and are called chaotropic. Salts in this category can also be used in the laboratory to help purify proteins that are being studied, by lowering their solubility and causing them to “salt out.”

see also Heavy Metal Toxins; Proteins.

Thomas A. Holme

Bibliography

Branden, C., and Tooze, J. (1998). Introduction to Protein Structures, 2nd edition. London: Taylor and Francis.

Creighton, T. E. (1993). Proteins, 2nd edition. New York: Freeman.

Fagain, C. O. (1997). Protein Stability and Stabilization of Protein Function. Georgetown, TX: Landes Bioscience.

Table of Contents

The process that causes a protein to lose its shape is known as denaturation. Denaturation is usually caused by external stress on the protein, such as solvents, inorganic salts, exposure to acids or bases, and by heat.

What are 3 factors that cause proteins to denature?

Changes in pH, Increased Temperature, Exposure to UV light/radiation (dissociation of H bonds), Protonation amino acid residues, High salt concentrations are the main factors that cause a protein to denature.

What is denaturation What causes it?

Denaturation defines the unfolding or breaking up of a protein, modifying its standard three-dimensional structure. Proteins may be denatured by chemical action, heat or agitation causing a protein to unfold or its polypeptide chains to become disordered typically leaving the molecules non-functional.

What happens during enzyme denaturation?

Enzyme structures unfold (denature) when heated or exposed to chemical denaturants and this disruption to the structure typically causes a loss of activity. Protein folding is key to whether a globular protein or a membrane protein can do its job correctly. It must be folded into the right shape to function.

What is denaturation and renaturation of protein?

The key difference between denaturation and renaturation of protein is that denaturation is the loss of native 3D structure of a protein while renaturation is the conversion of denatured protein into its native 3D structure. Therefore, denaturation is the process by which a protein loses its native 3D structure.

What is an example of denaturation?

Common examples When food is cooked, some of its proteins become denatured. This is why boiled eggs become hard and cooked meat becomes firm. A classic example of denaturing in proteins comes from egg whites, which are largely egg albumins in water. The same transformation can be effected with a denaturing chemical.

What 3 things can denature enzymes?

Enzyme activity can be affected by a variety of factors, such as temperature, pH, and concentration.

Can denaturation be reversed?

Reversing Denaturation It is often possible to reverse denaturation because the primary structure of the polypeptide, the covalent bonds holding the amino acids in their correct sequence, is intact. However, denaturation can be irreversible in extreme situations, like frying an egg.

What things change color when their proteins are denatured?

An egg white before the denaturation of the albumin protein causes the transucent substance to change in color and viscosity. The heat-caused denaturation in albumin protein in egg whites causes the once translucent, runny substance into one that is white and firm.

What does denaturation mean and why is it important?

Denature means lose their structure and unfold due to acid or temperature. hydrogen bonds in between amino acids are disrupted and falls apart because of that. Once its shape is messed up, it can’t do what it usually does. When it becomes denatured, it can’t break down the molecules and speed up the processes.

Is denaturation a chemical reaction?

Introduction. The term denaturation, as applied to proteins, typically refers to a loss of function. Chemical denaturation is a process by which this structure is destroyed through chemical means, via addition of denaturing agents (denaturants) to the solvent.

What happens to the body if enzymes are denatured?

Enzyme Functions and Denaturation Enzymes have specific functions in the body, such as working to break down food or causing other chemical processes. Enzymes never die, but they are not considered to be either living or nonliving organisms. When enzymes denature, they are no longer active and cannot function.

What are the 4 causes of protein denaturation?

Various reasons cause denaturation of protein. Some of them are an increased temperature that ruptures the protein molecules’ structure, changes in pH level, adding of heavy metal salts, acids, bases, protonation of amino acid residues, and exposure to UV light and radiation.

What activities can cause denaturation?

Note 2: Denaturation can occur when proteins and nucleic acids are subjected to elevated temperature or to extremes of pH, or to nonphysiological concentrations of salt, organic solvents, urea, or other chemical agents. Note 3: An enzyme loses its catalytic activity when it is denaturized.

What is denaturation of protein give example?

When a solution of a protein is boiled, the protein frequently becomes insoluble—i.e., it is denatured—and remains insoluble even when the solution is cooled. The denaturation of the proteins of egg white by heat—as when boiling an egg—is an example of irreversible denaturation.

Why is denaturation important?

The way proteins change their structure in the presence of certain chemicals, acids or bases – protein denaturation – plays a key role in many important biological processes. And the way proteins interact with various simple molecules is essential to finding new drugs.

Is denatured protein healthy?

Is denatured protein good or bad? You may have read that denatured protein is bad for you, and that you want to avoid denaturing your protein as much as possible. That’s not automatically true. Denaturing sounds awful, but all it means is breaking protein down from its original form.

Does water cause denaturation of proteins?

Proteins consist of one or more polypeptides, chains of amino acids held together by peptide bonds. If a protein in water is heated to temperatures approaching the boiling point of water, these chains will lose their structure and the protein will denature (unfold).

What is denaturation process?

Denaturation, in biology, process modifying the molecular structure of a protein. Denaturation involves the breaking of many of the weak linkages, or bonds (e.g., hydrogen bonds), within a protein molecule that are responsible for the highly ordered structure of the protein in its natural (native) state.

What conditions can denature a protein?

A wide variety of reagents and conditions, such as heat, organic compounds, pH changes, and heavy metal ions can cause protein denaturation.

Does salt cause enzyme denaturation?

If the salt concentration is close to zero, the charged amino acid side chains of the enzyme molecules will attract to each other. The enzyme will denature and form an inactive precipitate. An intermediate salt concentration such as that of human blood (0.9% ) or cytoplasm is the optimum for many enzymes.

Is protein denaturation reversible or permanent?

Protein denaturation is said to be irreversible when the denatured state achieved by increasing temperature or by using chemical denaturants is unable to return to the native, biologically functional state upon removal of the factor that caused denaturation.

How to denature a protein

Grade Level: High School; Type: Biochemistry

Objective:

The objective of this experiment is to determine whether all proteins denature at the same temperature.

Research Question:

  • What happens when a protein denatures?
  • Do all proteins denature at the same temperature?
  • What temperature does albumen denature at?
  • What temperature does keratin denature at?
  • What temperature does casein denature at?
  • Why might proteins denature at different temperatures?

Denaturation is a process in which proteins lose their structure when attacked by forces like a strong acid, heat, or a solvent like alcohol. If a protein is denatured, it can die. In this experiment, you will determine the temperature that will denature proteins like albumen, casein, and keratin. Eggs are mostly albumen, milk is largely casein, and hair is mostly keratin.

Materials:

  • Small saucepan
  • 6 eggs (any size)
  • 2 mixing bowl
  • Candy thermometer
  • Powered Milk
  • Cookie Sheet
  • Aluminium foil
  • Hair from a hairbrush
  • Comb
  • Toaster oven (or conventional oven)

Experimental Procedure

  1. Crack an egg over the first bowl and separate the yolk and white. Use two bowls, keeping all the whites in one of the bowls. Make sure that your yolks do not contaminate the whites.
  2. Transfer the whites into a small saucepan. Place the candy thermometer into the saucepan.
  3. Gently heat the whites. Record the temperature when their texture changes.
  4. Clean the saucepan, thermometer, and bowls.
  5. Make two cups of reconstituted powdered milk according to the package directions and add to the saucepan. Place the candy thermometer in the saucepan.
  6. Gently heat the milk. Record what temperature the texture of the milk changes or a skim forms over the top.
  7. Cover a cookie sheet with aluminum foil.
  8. Preheat the oven for ten minutes to 200 degrees.
  9. Using a comb, pull all the hair out of a hairbrush and put on the cookie sheet.
  10. Put the cookie sheet and hair into the oven. Let it heat up for 15 minutes. Inspect the hair for any changes.
  11. Increase the temperature by 25 degrees. After 15 minutes, inspect the hair again.
  12. Keep increasing the temperature in 25-degree increments. Note when the hair texture changes.

Terms/Concepts: protein, amino acid, denaturation

References:

  • Animation: Protein denaturation, Essentials of Anatomy and Physiology
  • Animation: Protein structure, Discover Biology

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my antibody can’t bind with my protein of interest during IP process, but the antibody can perfectly recognize it on western membrane. No matter how I change the binding condition the same thing keeps happening.

So now, i suspect that the native structure of protein is not accessible to antibody. Probably I need a condition in which protein of interest is in a denatured form but antibody is functional.

My plan is: I could denature the protein first by SDS, then reduce SDS concentration by dialysis, then add antibody. I guess I could reduce SDS concentration to 0.1%, which is still ok for antibody.

Think about SDS-PAGE, we denature protein, after which the proteins are in an environment that contains 0.1% SDS.

Will it work? I’m afraid that in solution, the protein will fold back when SDS concentration is reduced.

I need some advice from you
Thanks a lot!

I wouldn’t go with SDS, as it’s very difficult to dialyse away from protein. Once it binds, it’s unlikely to leave. Instead, I’d try guanidinium. Concentrate your protein as high as you can get without aggregates, then denature with 6M Guanidine-HCl. You then dilute that into a solution containing your antibody. If you’re set on SDS, you could do the same thing with dilution and not worry about refolding (antibody would have diffusion-limited access to unfolded protein until it either refolds or aggregates). Antibodies may even be stable in 6M Gu-HCl (anybody know?)

The downside with either method is that you may aggregate your protein of interest when you remove the denaturing agent. If you have access to a fluorometer, you can check for aggregates by measuring light scatter, if it’s important.

Out of curiosity, what’s the purpose of the IP? Are you trying to pulldown another interacting molecule, or is it a purification step? I ask because a denatured protein is not going to behave in the same way as the native form, so any interaction or activity beyond antibody binding could be lost.

Also, whether the protein refolds in solution is another matter entirely. It may be able to do so on its own, or it may require chaperones for proper folding.

One easy thing to try would be to boil the protein then cool quickly on ice. Then there is no need for dialysis or other chemicals which can interfere with antibody binding.

Thanks a lot to aludlam and ajames:)

Maybe I should post this topic on chromatin board
Actually I’m dealing with chromatin immunoprecipitation, and I got problem in protein purification step. In this case, I don’t care about the protein interacting partners or its native structure, I just want to purify the protein without releasing the DNA fragment bound on it.

As the protein-DNA crosslink will be reversed by heat, therefore I can’t boil the sample to denature protein.

I plan to incubate my protein sample in a denaturing environment, then dilute it so that when antibody is added in, it won’t die.

SDS? Gu-HCl? which one is better?

For your purposes, I’m guessing neither is optimal. SDS will coat the protein with a net negative charge, which could repel the DNA. GuHCl is a safer bet, but I don’t know whether it affects the ionic state of the protein, and the tertiary structure may be important for binding. Only way to find out is to try and see.

If you try guanidine, you could also try 8M Urea, which has similar effects but may be a bit gentler than Gu-HCl. Also, you may not need to go to the upper range of either of these reagents. I’ve gotten efficient denaturation of a model protein with 3M Gu-HCl, and some proteins are purified in the presence of 2M Urea.

SUCKASACCHARIDE welcome to suck a saccharide the most amazing biochemistry blog you will ever experience,it would be both funny and educational so hope you enjoy.

Ok guys so we did the different level of proteins and we are now going to discuss the denaturation of proteins and the factors that affects the different structure levels.

DENATURATION OF PROTEINS

The denaturation of proteins is defined as any non covalent changes in the structure of the protein.The change alter the secondary ,tertiary and quaternary structre of the molecules. The primary structure or the amino acid sequence remains the same after the denaturation process.

The most common factors that denatures proteins includes:

  • Heat: This disrupts hydrogen bonds and non-polar hydrophobic interactions.
  • Alcohol: This affects the hydrogen bonds that are formed between the amide groups of the secondary level and it also affects the hydroden bonding between the side chains of the tertiary level.
  • Acids and Bases and Heavy Metals: Strong acids and bases and heavy metals denatures the protein the same way by disrupting the salt bridge.
  • Urea : Destabalizes internal bonds and unfolds the protein because it (urea) is a chaotrophe.
  • UV : The effects are similar to heat
  • Organic solvents :The interupt the intracovalent interactions of proteins. example Acetone.
  • Detergents: Breaks up positive and negative interactions in the protein chains.

FACTORS THAT AFFECT THE DIFFERENT STRUCTURE LEVELS OF PROTEINS

After reading the denaturation of proteins you will realise that the primary structure is never affected, However it is very important because the amino acid sequence is what synthesizes what the structure of the protein would be either secondary, tertiary or quaternary.

So do you remember what level of protein you will find the alpha helix and beta pleated sheets. if you said secondary level you are right.

Glycine is the smallest amino acid to exist because it has Hydrogen as its “R” group.It is not allowed in the helix because it has high conformational flexibility meaning it moves around too much and destabalizes the alpha helix.

Proline forms a rigid ring, the nitrogen-carbon bond destabalizes the alpha helix. It is usually found at the end of an alpha helix because it alters the direction of the polypeptide chain and terminates the helix.

Large bulky side chains which simply means big “R” groups causes steric interferences and stops the formation of the helical structure

Interactions of the amino acids at the ends of the helix this means that dipoles lines up to form a macrodipole. As we know the “N” te rminal end is positive and the “C” terminal end is negative. Therefore to stabalize the alpha helix a negative amino acid residue will be placed at the “N” terminal end and a positive amino acid residue will be placed at the “C” terminal end.

The factors that affects the Tertiary and Quaternary structures includes:

HYDROPHOBIC EFFECT this is a spontaneous type of folding which allows the non-polar hydrophobic residues are on the interior of the structure and the polar hydrophilic residues are on the exterior of the chain hence the stability of the protein is maintained.

Electrostatic forces this deals with the attraction of opposite charges.

Hydrogen bonding the joining of a hydrogen atom with either an oxygen atom or a nitrogen atom.

Disulphide bonds are the oxidation ot two cysteine molecules.

Table of Contents

The way proteins change their structure in the presence of certain chemicals, acids or bases – protein denaturation – plays a key role in many important biological processes. And the way proteins interact with various simple molecules is essential to finding new drugs.

What does denaturation mean and why is it important?

Denature means lose their structure and unfold due to acid or temperature. hydrogen bonds in between amino acids are disrupted and falls apart because of that. Once its shape is messed up, it can’t do what it usually does. When it becomes denatured, it can’t break down the molecules and speed up the processes.

What is the purpose of denaturing proteins?

Denaturation, in biology, process modifying the molecular structure of a protein. Denaturation involves the breaking of many of the weak linkages, or bonds (e.g., hydrogen bonds), within a protein molecule that are responsible for the highly ordered structure of the protein in its natural (native) state.

Do proteins denature in our body?

As it happens, however, most of the proteins in your body begin to denature at 105.8 degrees. This means that if your body temperature goes over that value more than briefly, you are likely to notice degradation in an assortment of functions, and your condition may even become life-threatening.

Why is denaturation important in digestion?

As you have read, denaturing is an important part of the digestive process. This unfolds the proteins and makes the protein bonds more accessible by the enzymes so that proteins can be efficiently broken down. When you cook a protein, you denature it in a similar way to how the HCL in your stomach does.

What are some examples of denaturation?

When food is cooked, some of its proteins become denatured. This is why boiled eggs become hard and cooked meat becomes firm. A classic example of denaturing in proteins comes from egg whites, which are typically largely egg albumins in water. Fresh from the eggs, egg whites are transparent and liquid.

Is denaturation good or bad?

Denaturing sounds awful, but all it means is breaking protein down from its original form. You denature proteins when you digest them, and in some cases, buying denatured (think pre-digested) protein can help you absorb the amino acids better. A good example is hydrolyzed collagen.

How is denaturation used in hospitals?

The proteins in eggs denature and coagulate during cooking. Other foods are cooked to denature the proteins to make it easier for enzymes to digest them. Medical supplies and instruments are sterilized by heating to denature proteins in bacteria and thus destroy the bacteria.

What 3 things can denature proteins?

Proteins are denatured by treatment with alkaline or acid, oxidizing or reducing agents, and certain organic solvents. Interesting among denaturing agents are those that affect the secondary and tertiary structure without affecting the primary structure.

Why is denaturation a bad thing?

Because proteins’ function is dependent on their shape, denatured proteins are no longer functional. During cooking the applied heat causes proteins to vibrate. This destroys the weak bonds holding proteins in their complex shape (though this does not happen to the stronger peptide bonds).

What is denaturation of protein explain with example?

Denaturation of proteins is an irreversible change in which proteins get precipitated when they are heated with alcohol, concentrated inorganic acids or by salts of heavy metals. Protein is uncoiled and its shape is destroyed. Characteristic biological activity is lost.

What is food denaturation?

“Denature” means to “destroy the characteristic properties of (a protein or other biological macromolecule) by heat, acidity, or other effects that disrupt its molecular conformation” (Merriam-Webster). When food is denatured it refers to the physical change that occurs, mostly the proteins.

What 3 things can denature enzymes?

Enzyme activity can be affected by a variety of factors, such as temperature, pH, and concentration.

What is the difference between precipitation of a protein and its denaturation?

Denaturation and precipitation are in fact different things, even though they are often linked in protein chemistry. Denaturation is the loss of native conformation of a protein’s structure. So in most cases, denaturation of proteins causes their precipitation.

What things change color when their proteins are denatured?

An egg white before the denaturation of the albumin protein causes the transucent substance to change in color and viscosity. The heat-caused denaturation in albumin protein in egg whites causes the once translucent, runny substance into one that is white and firm.

Can denaturation be reversed?

Reversing Denaturation It is often possible to reverse denaturation because the primary structure of the polypeptide, the covalent bonds holding the amino acids in their correct sequence, is intact. However, denaturation can be irreversible in extreme situations, like frying an egg.

What happens if the shape of a protein is altered?

Because form determines function, any slight change to a protein’s shape may cause the protein to become dysfunctional. Small changes in the amino acid sequence of a protein can cause devastating genetic diseases such as Huntington’s disease or sickle cell anemia.

What do you mean by protein denaturation?

Protein denaturation is the net effect of alterations in the biological, chemical, and physical properties of the protein by mild disruption of its structure.

Does water cause denaturation of proteins?

Proteins consist of one or more polypeptides, chains of amino acids held together by peptide bonds. If a protein in water is heated to temperatures approaching the boiling point of water, these chains will lose their structure and the protein will denature (unfold).

What can cause denaturation?

The process that causes a protein to lose its shape is known as denaturation. Denaturation is usually caused by external stress on the protein, such as solvents, inorganic salts, exposure to acids or bases, and by heat.

What is denaturation milk?

For example, milk consists of a variety of nutrients, including about 3% proteins. When introducing an acid, the ionic interactions between the casein phosphate groups and calcium ions are disrupted, causing the casein proteins to denature.

How does denaturation affect digestion?

Protein denaturation can also alter gastric emptying of the protein, consequently affecting digestive kinetics that can eventually result in different post-prandial plasma amino acid appearance. Apart from processing, the kinetics of protein digestion depend on the matrix in which the protein is heated.

What is the biological effect of denaturation of proteins?

During denaturation of proteins, the secondary and tertiary structures get destroyed and only the primary structure is retained. Covalent bonds are broken and interaction between amino-acid chains gets disrupted. This results in the loss of biological activity of the proteins.

Ultraviolet absorption spectroscopy of proteins

How to denature a protein

Ultraviolet microscope image of a protein crystal in solution. The crystal is dark as it absorbs UV light while the solution does not, resulting in a bright background.

How to denature a protein

Jablonski Energy Level Diagram Depicting Absorption and Fluorescence Transitions

How to denature a protein

Proteins, such as those in animal tissue and plants, strongly absorb ultraviolet (UV) light at approximately 280 nm. Rather, it is some of the amino acids which make up the proteins that absorb the UV light. The strong absorption of UV light by proteins allows for rapid detection and identification of protein samples, both liquid and solid, by microscopy and microspectroscopy.

Amino Acids

Commonly, the optical absorption of proteins is measured at 280 nm. At this wavelength, the absorption of proteins is mainly due to the amino acids tryptophan, tyrosine and cysteine with their molar absorption coefficients decreasing in that order. Of course, the molar absorption coefficient of the protein itself at 280 nm will depend upon the relative concentrations of each of these three amino acids. Therefore, different proteins can have different absorption coefficients and even the wavelength of the maximum absorption may differ. This fact can be used to help identify different types of proteins by relatively fast and simple optical tests.

Imaging Proteins by UV Absorbance

Most commonly, protein crystals are imaged by their intrinsic protein fluorescence. This is mostly the fluorescence of tryptophan. As such, protein fluorescence requires very powerful UV light sources and very sensitive cameras because the fluorescent emission from proteins is so weak. However, powerful UV light sources can destroy the protein due to long exposure times required to obtain significant data.

A much faster way to image proteins, either in cells, tissues or as crystals, is to utilize their strong absorption of UV light as a contrasting mechanism. By using a ultraviolet microscope or microspectrophotometer equipped for UV imaging, the sample containing the protein is imaged with 280 nm light. The protein will absorb this light more strongly than the surrounding sample and will appear darker. See the picture above for an example of UV absorption of a protein crystal in salt solution. This technique is very fast, exposing the protein to UV light for far less time.

Spectroscopy of Proteins by UV Absorption

CRAIC Technologies microspectrophotometers are used to acquire spectra of microscopic samples containing proteins, such as individual protein crystals, by their UV absorption. The microspectrophotometer consists of a UV-visible-NIR range microscope integrated with a spectrophotometer. As such, it is able to measure the UV-visible-NIR spectra of microscopic samples of tissue, protein crystals and other protein containing structures. By using absorption, it is able to measure these samples quickly and non-destructively.

Microspectroscopy allows the user to learn more about the optical features and the chemical structure of the protein. Additionally, microspectroscopy also allows for the determination of the concentration of protein in a sample as the absorption at 280 nm is proportional to the protein concentration.

If the protein sample does not have tryptophan or tyrosine, both of which absorb at 280 nm, the concentration can still be easily measured by the Scopes Method. In this particular method, the protein concentration is determined by the absorption at 205 nm in which the peptide bonds are analyzed directly.

DNA or RNA purity can also be determined by measuring the absorption ratios of 260 to 280 nm. This is because the nucleic acids that make up DNA and RNA absorb strongly at 260 nm. A ratio of about 2.0 is considered “pure” for RNA while a ratio of about 1.8 is considered “pure” for DNA. Lower ratios indicate the presence of protein.

Summary

Proteins absorb strongly at 280 nm due to three types of its constituent amino acids. The peptide bonds found in the amino acids also absorb at 205 nm. The UV absorption of protein can be used both to quickly image and acquire spectra of microscopic samples non-destructively. The spectra can also be used to determine protein concentrations and the relative amounts of protein to DNA or RNA.

Proteins are large, organic – in the science sense of organic, not the food marketing sense – molecules that help us to convert food into energy, supply oxygen to our blood and muscles and drive our immune systems.

Proteins consist of one or more polypeptides, chains of amino acids held together by peptide bonds. If a protein in water is heated to temperatures approaching the boiling point of water, these chains will lose their structure and the protein will denature (unfold).

A more clear example of denaturing/unfolding occurs when an egg is hard-boiled: the structures of the proteins in the egg unfold with temperature and stick together creating a solid. In the egg’s case, the process cannot be reversed but there are many examples where cooling the protein results in refolding of the structure. Proteins also evolved in a water-rich environment so it is generally accepted that they are dependent on water to survive and function, like in the refolding process, however some new findings suggest this isn’t necessarily the case.

Using circular dichroism, a spectroscopic technique, Dr. Adam Perriman of Bristol’s School of Chemistry and colleagues have shown that the oxygen-carrying protein myoglobin can refold in an environment that is almost completely devoid of water molecules.

Perriman said, “We achieved this by attaching polymer molecules to the surface of the protein and then removing the water to give a viscous liquid which, when cooled from a temperature as high as 155°C, refolded back to its original structure. We then used the Circular Dichroism beamline (B23) at Diamond Light Source, the UK’s national synchrotron science facility in Oxfordshire, to track the refolding of the myoglobin structure and were astounded when we became aware of the extremely high thermal resistance of the new material.”

The results could pave the way for the development of new industrial enzymes where hyper-thermal resistance would play a crucial role, in applications ranging from biosensor development to electrochemical reduction of CO2 to liquid fuels.

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One of the most important scientific arguments in favor of totally raw diets, and uncooked foods in general, is that the proteins in foods are denatured by the high temperatures of cooking.
Unfortunately, this powerful concept is obscured and confused by an ancient book by Edward Howell, titled Enzyme Nutrition, wherein he speculates that: “I adhere to the philosophy [Note: philosophy, not science – ljf] that both the living organism and its enzymes are inhabited by a vital principle or life energy which is separate and distinct from caloric energy”. “The enzyme complex harbors a protein carrier inhabited by a vital energy factor.”, yet he can not demonstrate any existence of this mysterious “life energy”, nor describe how to detect and measure it. This unsupportable metaphysical concept was actively propagated by the old Hippocrates Health Institute in Boston in the 1970’s and it still plagues and embarrasses the raw food community to this very day.
How do we know if something is alive? The best test I have come up with is that it must eat, excrete, and reproduce. Another quality might be the ability to self-repair. Clearly, enzymes, which are merely proteins, do not manifest any of these properties unique to living beings.
Enzyme Nutrition still sells, and holds the Amazon.com Sales Rank: 29,980 as of June, 2003.
The back cover of the book states: “In 1930, Dr. Howell established his own facility for the treatment of chronic ailments, . ” and he retired in 1970, so, clearly, his concepts are over 75 years out of date! If information doubles every ten years, there is now available

180 times as much information today as when Howell made these speculations. Certainly, IF enzymes were really “alive”, it would be well known today.
He further claims: “. the capacity of living organisms to make enzymes . is limited and exhaustible” quite to the contrary of modern biochemistry.
The claim that the enzymes in “foods” help in the foods’ own digestion is nonsensical, as plant enzymes are quite specific to individual chemical reactions supporting the plant’s biochemistry, and they certainly do not “know” anything about human digestive biochemistry and they have not changed their biochemistry for our digestive convenience. Since plant species tend to be enormously older than our little psychotic ape species, just how did the plants anticipate human evolution and graciously alter their own biochemistry for our convenience?
Further, enzymes generally operate within very limited ranges of temperatures and pH (acidity) and since the human stomach tends to be very acidic, whereas plant sap is not, plant enzymes would not be active in the human stomach. Finally, enzymes are proteins, so wouldn’t they be digested the same as other food proteins, thus destroying their enzymatic action?

So, what does the heating and consequent denaturing of proteins really do?
Proteins are composed of strings of amino acids arranged in quite specific orders, like beads on a string . This linear structure is the primary structure. However, these strings of amino acids are then folded upon themselves by relatively weak hydrogen bonds (proton bonds) into complex three-dimensional structures having secondary, tertiary, and quaternary “higher order” structures. The biological activity of molecules is determined by these higher three-dimensional structures and how they physically fit into the three-dimensional structures of other biomolecules. Since strict physical conformance of the enzyme to the food protein it acts upon (say, in digesting it) must be satisfied for the bioactivity, this tight complementary relationship is frequently referred to as a “lock and key” model. Destroy these higher structures and the chemical most probably becomes biologically inactive: bend the key and it will not open the lock. Denaturation is a process that alters a protein’s native conformation and biological activity. If the tertiary or quaternary structure of a protein is altered, e.g., by such physical factors as extremes of temperature, changes in pH, or variations in salt concentration, the protein is said to be denatured; it usually exhibits reduction or loss of biological activity.
[A good primer on protein folding, and an opportunity to donate some of your unused computer cycles to a research project.]

Lehninger, Biochemistry, 2ed., 6th printing, 1981, p. 144

Here, we see that the higher structures of the protein in question collapse at

The Top Methods for DNA Denaturation

How to denature a protein

How to denature a proteinDNA denaturation is the process of breaking down the DNA molecule, generally for the purposes of comparison or sequencing. As with many laboratory techniques, there are a variety of ways to denature DNA — and each of them tend to be better for specific applications. The top three methods of DNA denaturation are heat, NaOH treatment, and salt. Each of these methods will break the bonds between strands, but may do so with a greater degree of accuracy or lessened disruption.

DNA Denaturation through Heat

DNA can be denatured through heat in a process that is very similar to melting. Heat is applied until the DNA has unwound itself and separated into two single strands. Once the strands have been separated, the DNA will then be cooled back down to a stable temperature. The process of cooling allows molecules to be formed in the DNA mixture, which then produces certain sequences that can be looked at as markers. Heat denaturation is frequently used when comparing the differences between species. Though DNA melting is a fairly simple and straightforward process, it is generally not used when accuracy is required. Heated DNA denaturation is considered to be less accurate than DNA sequencing and is used for more broad scope applications. This type of denaturation may also be used within the polymerase chain reaction.

DNA Denaturation through NaOH Treatment

Apart from heating, chemical denaturation can also be achieved through the use of NaOH. A certain concentration of NaOH can be used to denature DNA safely, often in as little as one minute. As the concentration of NaOH used is reduced, the process will take longer — but the DNA can still be fully denatured. NaOH has been shown to be one of the most effective and reliable methods of complete denaturing. Other chemicals, such as formamide, are not able to denature DNA as rapidly or as reliably. Because NaOH can be used at a variety of concentrations, it is also able to be scaled quite easily. Furthermore, the DNA that is denatured with NaOH can be renatured through the use of a phosphate buffer. DNA that is denatured through other chemicals, such as DMSO, are not able to be fully renatured in this fashion — and this can lend NaOH to more applications.

DNA Denaturation through Salt

A high concentration of salt will cause DNA to naturally denature, given the right concentration of salt. DNA denaturation with salts are similar to denaturation through the use of organic solvents. In general, DNA denaturation through salt cannot be renatured. Salt is often used in addition to an acid for the full denaturation of DNA, and it may also be used in conjunction with heat. Salt is not usually used as the sole process of denaturation — it’s usually used alongside other chemicals such as isopropanol and ethanol. This process is able to be used on larger volumes of DNA, which makes it less useful for highly accurate and specific work, but more useful for scaling up and processing DNA in larger quantities.

Though there are many techniques associated with DNA denaturation, the end result is the same: the bonds between the strands are broken and new molecules are formed, which can then be compared as desired. The ideal process of DNA denaturation depends on what the DNA needs to be used for, how accurate and specific the comparisons need to be, and the volume of material that has to be processed. In general, both heat and salt denaturation can be easily scaled and used with larger quantities, while NaOH denaturation may be slightly more accurate and useful in smaller quantities.

Cooking with chemistry

In class today we made a baked custard tart. The two functional properties of protein we looked at during this practical was denaturation and coagulation.

Blind baked pastry

DENATURATION: occurs when the bonds holding the helix shape are broken and the strands of the helix separate and unravel. It is a permanent change in the structure of proteins. The functional property of protein, denaturation is useful in food preparation for example whisking eggs which is a component of many food products, the marinating a piece of meat where the acid tenderises the meat before cooking, making sour cream and yoghurt.

COAGULATION: Is more visible then denaturation, this process occurs when denatured proteins separate from other nutrients and solidify or semi soldify. Applying heat for a long period of time will cause the protein structure to create a network and trap liquid which will form a gel. Coagulation is used in food preparation most commonly for cooking eggs, some examples include; raw eggs being cooked eg boiled or scrambled as part of a dish, making a quiche with coagulated eggs, meringue (denaturation for beating the eggs, coagulation for cooking the egg product), pretty much any egg product being cooked. Also cooking meats like chicken, and the process of making cheese.

Difference between denaturation and coagulation:

  • Denaturation happens before coagulation
  • coagulation is more visible then denaturation
  • coagulation uses denatured proteins
  • you can over coagulate, but cant over denature

The physical changes that occurred throughout the making of the custard tart is the mixing ingredients together for both the pastry and the custard. The rolling out of the dough to make a smooth thin pastry and pricking the bottom of the pastry to allow steam to escape

Custard before heated

The main sources of protein found in the custard was the egg and the milk. Both the egg and the milk are globular proteins. Globular proteins are strands of proteins that are twisted into a rounded, compact shape. The type of globular found in the eggs, or more specifically the egg whites is albumin. The main type of globular protein found in the milk is alpha lacto globulin and beta lacto globulin.

In the making of the custard, then process of denaturation and coagulation occurred. When denaturation is occurring the bonds holding the helix shape are broken and unravel. When coagulation is occurring the unravelled protein strands begin to re-join with other strands forming a solid mass. Egg was the food mainly responsible for this. For this change to occur several factors were present for this to occur. Some factors include temperature, acidity, agitation and sugar. Heat causes proteins to denature or unravel. Proteins will denature and coagulate quicker in higher temperatures, but different temperatures will affect the processes properties of different foods. Acid will help proteins denature. Denaturation occurs when the acid begins to break the bonds between strands of amino acids. Acid is used for thickening of dairy products. Agitation causes the protein strands to stretch, if there is too much mixing the strands are stretched too much to the point the protein is denatured which affects the function of the food. Finally sugar, in this case caster sugar, with proteins will mean the product will need a higher temperature before denaturation or coagulation occurs. The custard requires a higher temperature to coagulate the protein in the milk and thicken eggs due to the sugar. Other factors that affect denaturation and coagulation include enzymes and salt.

At 63 degrees Celsius egg whites begin to thicken, at 65 degree Celsius egg whites become a tender solid since coagulation has occurred. Eggs will continue to over coagulate as water is pushed out from between protein molecules. Between 63 degrees Celsius and 65 degrees Celsius is the optimum temperature for coagulation of eggs whites, the egg yolks require a slightly higher temperature to coagulate up to 70 degrees celsius.

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How to denature a protein

Protein precipitation is a method used to extract and purify proteins held in a solution. Large, complex molecules, proteins generally have parts that have a negative electrical charge and parts that have a positive charge, as well as hydrophilic and hydrophobic parts. There is a tendency for proteins in solution to clump together and precipitate out due to the attraction between the negatively and positively charged parts of the molecules and the mutual attraction of the hydrophobic parts. Counteracting this tendency, however, is the fact that in an aqueous solution, water molecules, which are polar, will tend to arrange themselves around the protein molecules due to the electrostatic attraction between oppositely charged parts of the water and protein molecules. This results in the protein molecules being kept apart and remaining in solution, but there are various methods for achieving precipitation of proteins.

The most commonly used method of protein precipitation is by adding a solution of a salt, a technique often referred to as “salting out.” The salt most frequently used is ammonium sulfate. The interaction of the salt ions with water molecules removes the water barrier between protein molecules, allowing the hydrophobic parts of the protein to come into contact. This results in the protein molecules aggregating together and precipitating out of solution. As a general rule, the higher the molecular weight of the protein, the lower the concentration of the salt that is required to cause precipitation, so it is possible to separate a mixture of different proteins in solution by gradually increasing the salt concentration, so that different proteins precipitate at different stages, a process known as fractional precipitation.

The solubility of a protein in an aqueous medium can be reduced by introducing an organic solvent. This has the effect of reducing the dielectric constant, which in this context can be regarded as a measure of the polarity of a solvent. A reduction in polarity means there is less of a tendency for solvent molecules to cluster around those of the protein, so that there is less of a water barrier between protein molecules and a greater tendency toward protein precipitation. Many organic solvents interact with the hydrophobic parts of protein molecules, causing denaturization; however, some, such as ethanol and dimethyl sulfoxide (DMSO), do not.

Although proteins can have negatively and positively charged parts, often, in solution, they will have an overall positive or negative charge that varies according to the pH, and keeps them apart through electrostatic repulsion. In acidic conditions, with a low pH, proteins tend to have an overall positive charge, while at high pH, the charge is negative. Proteins have an intermediate point at which there is no overall charge — this is known as the isoelectric point and for most proteins, it lies in the pH range 4-6. The isoelectric point for a dissolved protein can be reached by adding an acid, usually hydrochloric or sulfuric acid, to reduce the pH to the appropriate level, allowing clustering and precipitation of the protein molecules. A disadvantage of this method is that the acids tend to denature the protein, but it is often used to remove unwanted proteins.

Other methods of protein precipitation include non-ionic hydrophilic polymers and metal ions. The former reduce the amount of water available to form a barrier between protein molecules, allowing them to clump together and precipitate. Positively charged metal ions can bond with negatively charged parts of the protein molecule, reducing the tendency of the protein to attract a layer of water molecules around it, again allowing the protein molecules to interact with one another and precipitate out of solution. Metal ions are effective even in very dilute solutions.

Through the process of cooking, molecular transformations alter the macroscopic properties of our food. Consider what happens when you fry an egg: the transparent, liquid egg whites become an opaque white solid. These striking changes in the egg’s color and texture are a result of protein denaturation.

A raw egg white is essentially a suspension of proteins in water. These proteins are made of long chains of amino acids that “fold” into specific and stable three-dimensional structures. Heating the proteins in an egg white causes them to “unfold” and no longer maintain their inherent structure. In this state, proteins are said to be denatured and will readily stick to one another to form an extensive protein network.

How to denature a protein

The formation of such denatured protein networks underlies many of the macroscopic changes we observe during the cooking of protein-rich foods such as eggs and meat. The extent of protein network formation affects the final texture of cooked meat and eggs. A steak cooked medium-well, for example, will have a denser protein network that gives rise to a tougher steak than one that is cooked medium-rare [1]. Denatured protein networks also affect the optical properties of protein-rich foods. Because a cooked protein network scatters light more effectively than a suspension of uncooked proteins, eggs and fish become more opaque as they are cooked [2].

We usually associate cooking with heat-related processes, but there are other ways to denature proteins. For example, you can “cook” an egg in acetic acid:

This form of chemical cooking relies on high salt concentrations or extreme pH conditions to denature proteins and “cook” food. Although an egg “cooked” in pure acetic acid may not have broad taste appeal, chemical cooking is used to prepare a variety of edible dishes including brined salmon (lox), pickled herring, and lutefisk.

In this recipe for ceviche, we will use an acidic (low pH) marinade to “cook” fish without heat.


Ingredients

1 pound of previously frozen* fish, 1 inch dice.
1 cup lime juice
1 cup diced tomatoes
1/2 cup diced red onion
1/2 cup roughly chopped cilantro
Kosher salt, to taste
1 avocado, sliced

*As a safety precaution, only use fish that has been previously frozen, as freezing reduces the risk of exposure to parasites in seafood [3].


Procedure

1. In a non-reactive dish, such as a glass bowl, toss together the fish, lime juice, and garlic.

2. Marinate for 30 minutes on ice or in the refrigerator.

Lime juice contains high concentrations of natural acids like citric acid and ascorbic acid. The pH of lime juice is around 2.5—more acidic than vinegar and similar to the pH of stomach acid. As the lime juice diffuses into the fish, its low pH will cause the proteins in the fish to denature and form protein networks. As a result, the fish will become tougher and more opaque.

Believe it or not, you can actually overcook your fish with lime juice! Leaving it too long will not only make it tough and dry, but it will also break down the connective tissue, causing your fish to fall apart (more on this from The Food Lab).

3. Once marinated, add the tomatoes, onion, cilantro, and salt to the fish.

Tomatoes provide extra acidity, but by now your fish should already be “cooked.”

4. Garnish with slices of avocado and eat with tortilla chips.


On
line Resources

  1. Recipe adapted from Keith Famie’s Ceviche
  2. The Food Lab: Ceviche and the Science of Marinades
  3. Heat changes protein structure: frying an egg animation

References Cited

  1. Bouton PE, Harris PV (1972) THE EFFECTS OF COOKING TEMPERATURE AND TIME ON SOME MECHANICAL PROPERTIES OF MEAT. Journal of Food Science 37: 140–144. doi:10.1111/j.1365-2621.1972.tb03404.x
  2. Anfinsen CB (1995) Advances in protein chemistry. Volume 47. San Diego; London: Academic Press. p. Available:http://site.ebrary.com/id/10240101. Accessed 20 January 2013.
  3. US Food and Drug Administration (2012) Food Facts: Fresh and Frozen Seafood: Selecting and Serving it Safely. Available:http://www.fda.gov/food/resourcesforyou/consumers/ucm077331.htm. Accessed 21 January 2013.

About the author: Liz Roth-Johnson is a Ph.D. candidate in Molecular Biology at UCLA. If she’s not in the lab, you can usually find her experimenting in the kitchen.

Through the process of cooking, molecular transformations alter the macroscopic properties of our food. Consider what happens when you fry an egg: the transparent, liquid egg whites become an opaque white solid. These striking changes in the egg’s color and texture are a result of protein denaturation.

A raw egg white is essentially a suspension of proteins in water. These proteins are made of long chains of amino acids that “fold” into specific and stable three-dimensional structures. Heating the proteins in an egg white causes them to “unfold” and no longer maintain their inherent structure. In this state, proteins are said to be denatured and will readily stick to one another to form an extensive protein network.

How to denature a protein

The formation of such denatured protein networks underlies many of the macroscopic changes we observe during the cooking of protein-rich foods such as eggs and meat. The extent of protein network formation affects the final texture of cooked meat and eggs. A steak cooked medium-well, for example, will have a denser protein network that gives rise to a tougher steak than one that is cooked medium-rare [1]. Denatured protein networks also affect the optical properties of protein-rich foods. Because a cooked protein network scatters light more effectively than a suspension of uncooked proteins, eggs and fish become more opaque as they are cooked [2].

We usually associate cooking with heat-related processes, but there are other ways to denature proteins. For example, you can “cook” an egg in acetic acid:

This form of chemical cooking relies on high salt concentrations or extreme pH conditions to denature proteins and “cook” food. Although an egg “cooked” in pure acetic acid may not have broad taste appeal, chemical cooking is used to prepare a variety of edible dishes including brined salmon (lox), pickled herring, and lutefisk.

In this recipe for ceviche, we will use an acidic (low pH) marinade to “cook” fish without heat.


Ingredients

1 pound of previously frozen* fish, 1 inch dice.
1 cup lime juice
1 cup diced tomatoes
1/2 cup diced red onion
1/2 cup roughly chopped cilantro
Kosher salt, to taste
1 avocado, sliced

*As a safety precaution, only use fish that has been previously frozen, as freezing reduces the risk of exposure to parasites in seafood [3].


Procedure

1. In a non-reactive dish, such as a glass bowl, toss together the fish, lime juice, and garlic.

2. Marinate for 30 minutes on ice or in the refrigerator.

Lime juice contains high concentrations of natural acids like citric acid and ascorbic acid. The pH of lime juice is around 2.5—more acidic than vinegar and similar to the pH of stomach acid. As the lime juice diffuses into the fish, its low pH will cause the proteins in the fish to denature and form protein networks. As a result, the fish will become tougher and more opaque.

Believe it or not, you can actually overcook your fish with lime juice! Leaving it too long will not only make it tough and dry, but it will also break down the connective tissue, causing your fish to fall apart (more on this from The Food Lab).

3. Once marinated, add the tomatoes, onion, cilantro, and salt to the fish.

Tomatoes provide extra acidity, but by now your fish should already be “cooked.”

4. Garnish with slices of avocado and eat with tortilla chips.


On
line Resources

  1. Recipe adapted from Keith Famie’s Ceviche
  2. The Food Lab: Ceviche and the Science of Marinades
  3. Heat changes protein structure: frying an egg animation

References Cited

  1. Bouton PE, Harris PV (1972) THE EFFECTS OF COOKING TEMPERATURE AND TIME ON SOME MECHANICAL PROPERTIES OF MEAT. Journal of Food Science 37: 140–144. doi:10.1111/j.1365-2621.1972.tb03404.x
  2. Anfinsen CB (1995) Advances in protein chemistry. Volume 47. San Diego; London: Academic Press. p. Available:http://site.ebrary.com/id/10240101. Accessed 20 January 2013.
  3. US Food and Drug Administration (2012) Food Facts: Fresh and Frozen Seafood: Selecting and Serving it Safely. Available:http://www.fda.gov/food/resourcesforyou/consumers/ucm077331.htm. Accessed 21 January 2013.

About the author: Liz Roth-Johnson is a Ph.D. candidate in Molecular Biology at UCLA. If she’s not in the lab, you can usually find her experimenting in the kitchen.

This article was co-authored by Courtney Fose, RD, MS and by wikiHow staff writer, Jessica Gibson. Courtney Fose is a Registered Dietitian and Certified Nutrition Support Clinician at the University of Arkansas for Medical Sciences. She has worked as a Dietitian since 2009, and received her MS in Clinical Nutrition from the University of Arkansas in 2016.

There are 15 references cited in this article, which can be found at the bottom of the page.

This article has been viewed 42,983 times.

Protein is one of the body’s most important nutrients, performing a variety of tasks in our bodies, including acting as enzymes and hormones (including insulin). The recommended dietary allowance (RDA) of protein defines the amount needed for the average healthy person and is suitable for roughly 97% of the population. [1] X Trustworthy Source Harvard Medical School Harvard Medical School’s Educational Site for the Public Go to source The amount of protein you need every day depends on your personal calorie needs, taking into account your age, sex, state of overall health, activity level and whether you need to lose or gain weight. Calculating the right amount of protein is important because too much protein can cause health problems. Excess protein can stress and overload the kidneys, be converted into body fat, cause dehydration and possibly increase the risk of diabetes, kidney disease and prostate cancer. [2] X Trustworthy Source Cleveland Clinic Educational website from one of the world’s leading hospitals Go to source

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Table of Contents

How do proteins denature western blot?

To denature, use a loading buffer with the anionic detergent sodium dodecyl sulfate (SDS), and boil the mixture at 95–100°C for 5 min. Heating at 70°C for 5–10 min is also acceptable and may be preferable when studying multi-pass membrane proteins.

What is the western blot technique?

A western blot is a laboratory method used to detect specific protein molecules from among a mixture of proteins. This mixture can include all of the proteins associated with a particular tissue or cell type. Following separation, the proteins are transferred from the gel onto a blotting membrane.

Why is SDS used in Western blotting?

SDS is generally used as a buffer (as well as in the gel) in order to give all proteins present a uniform negative charge, since proteins can be positively, negatively, or neutrally charged. The gel electrophoresis step is included in western blot analysis to resolve the issue of the cross-reactivity of antibodies.

What can I use instead of a western blot?

ProteinSimple™ has developed Simple Western™ assays for protein sizing and quantitative immunodetection as an alternative to traditional Western blot analysis. Assays are performed on Simon™, an instrument that integrates and automates all manual operations associated with Western blotting.

How much protein should I add to Western blot?

Make sure you load at least 20–30 µg protein per lane, use protease inhibitors, and run the recommended positive control.

Does RIPA buffer denature proteins?

RIPA buffer contains SDS, which is considered to be a harsh detergent; it can denature many proteins. C2978 is a buffered solution (pH 7.6) that contains a mild detergent; it is non-denaturing for most proteins.

How long does a Western blot take?

The Lyme disease blood test, western blot is used to detect antibodies specific for B burgdorferi. Preparation: No special preparation required. Test Results: 7-10 days. May take longer based on weather, holiday or lab delays.

What is a simple western?

The Simple Western is a reinvention of the entire Western blot, automating all steps from protein loading and separation, immunoprobing, washing, detection and quantitative analysis of data, finally giving researchers a complete, walk-away solution.

How is protein denatured in the human body?

Denaturing of meat proteins. Usually we eat meat protein that’s already denatured by heat – like cooked meat. The two parts of protein that are denatured in this process are collagen, which is the connective tissue that separates the bundles of muscle fibers, and the proteins inside the muscle fibers themselves.

What’s the procedure for denaturation of milk proteins?

Procedure for Milk (Casein) Denaturation: 1. Place 3 Teaspoons (

15 ml) each of milk into two cups. 2. Place 1 Teaspoon (5ml) of Lemon Juice in one of the cups containing the milk and stir. 3. Record Observations on the Milk Table. Adapted from: http://www.education.com/science-fair/article/denaturing-proteins/

What happens to avidin when protein is denatured?

This property of avidin is removed upon denaturing/cooking of the egg protein. Heat-denatured egg proteins are digested at about 90 percent in the human body whereas raw (undenatured) eggs are digested at only about 50 percent ( 9 ).

From my understanding, if you add lemon juice or apple cider vinegar to chicken soup, the acidic ingredients will denature the proteins.

If I want the protein in the soup to be denatured using these ingredients, what is the best way to go about it and get manximum denaturation and depth of penetration? Eg. should I marinade it, should I simply slow cook it with ingredients added at start, or should I brine in a cider/lemon solution? Please post the recipe I need.

Is it true when using a marinade, like salt, lemon and cider vinegar will only penetrate the surface?

What other ingredients can I use besides lemon and apple cider to do this job, or are lemon and cider vinegar the most effective?

1 Answer 1

When meat proteins get denatured excessively my the marinade, a cook would normally call that a failure (the texture is generally considered undesirable). So we’re not really experts in causing it—we try to avoid it!

For any substantial effect, the acidity has to be pretty high. Adding a little acid to a soup won’t do it. You need to add enough to bring the pH fairly low; your soup will be as sour as lemon juice. You probably don’t want this, so instead marinate.

When you put meat in marinade, only the outside of the meat is exposed to the marinade. So penetration starts from the outside. It slowly moves inward, but of course marinading time is limited, even in the fridge you can’t go too long to prevent spoilage (and freezing will stop the marinade from penetrating). You can add enzymes (papain, also known as “meat tenderizer”) to help. But it still has to get through from outside in.

To get full penetration, you want little distance from edge to middle as possible—that is, as little “inside” as possible. You could:

  • cut raw chicken into small chunks
  • cut raw chicken into thin slices
  • run raw chicken through a meat grinder, or buy ground chicken
  • run raw chicken through a food processor, etc.