Chem Explorers

Dissecting Disulfide Bonds: Their Formation Types and Breaking Methods

Proteins and Disulfide Bonds

1. Disulfide Bond Formation

Proteins are complex macromolecules that perform various essential functions in our body, such as catalyzing biochemical reactions, transmitting signals, and providing structure. The unique properties of different proteins are largely due to their three-dimensional structures.

One critical aspect of protein structure is the formation of disulfide bonds. Disulfide bonds are covalent bonds formed between two cysteine residues, and they play a crucial role in stabilizing protein structure.

Disulfide bond formation occurs when two thiol (-SH) groups on cysteine residues react chemically to form a covalent bond. This procedure occurs via an oxidation reaction that involves the transfer of electrons from one cysteine residue to another. The oxidized form of cysteine, known as cystine, forms the disulfide bond.

Disulfide bond formation is critical during protein folding, where it stabilizes the three-dimensional structure of the protein. In doing so, it ensures that the protein can perform its biological function.

2. Types of Disulfide Bonds

Disulfide bonds can also be classified into two types based on the orientation of the cysteine residues involved. In intramolecular disulfide bonds, the sulfur atoms involved in the bond are present within the same protein molecule.

These bonds contribute to the tertiary structure of a protein, and their formation often drives the protein-folding process. In contrast, intermolecular disulfide bonds form between two protein molecules and contribute to the quaternary structure of protein complexes.

Disulfide bonds can also be categorized into polar and nonpolar disulfide bonds. Polar disulfide bonds involve the formation of a bond between two cysteine residues that are hydrophilic or polar. These polar residues are usually present on the surface of proteins, where they interact with water. Nonpolar disulfide bonds involve the formation of a bond between two cysteine residues that are hydrophobic or nonpolar. These nonpolar residues are typically buried within the protein’s interior, away from water.

3. Functions of Disulfide Bonds

Disulfide bonds have various functions in biology. One of their main roles is to stabilize protein structure. By covalently linking cysteine residues, they prevent the protein from unfolding under various cellular conditions that may cause denaturation.

Disulfide bonds also play a role in redox processes, where they transfer electrons between proteins in reactions such as photosynthesis and respiration. This transfer of electrons is critical in the transmission of signals and generation of ATP, the energy currency of the cell.

Another function of disulfide bonds is in the folding of proteins. Protein folding is a complex process that often requires molecular chaperones, enzymes, and other proteins. Disulfide bonds play a crucial role in this process by providing a template for protein folding. They also act as checkpoints, ensuring that the protein has folded correctly, and preventing the misfolding that can be harmful to the cell.

Breaking Disulfide Bonds

1. Breaking Disulfide Bonds by Oxidation

Disulfide bonds are strong covalent bonds that help proteins maintain their three-dimensional structures. Nonetheless, there are situations when researchers may want to break these bonds to study the protein’s structure and function.

The most common way to break disulfide bonds is by using reducing agents. Examples of reducing agents include -mercaptoethanol and dithiothritol (DTT). These agents contain sulfhydryl (-SH) groups that can react with the oxidized disulfide bonds in proteins, reducing them back to cysteine residues.

This reduction reaction liberates the protein from the disulfide bond, resulting in the unfolding of the protein. Researchers often use reducing agents to break disulfide bonds when they want to study a protein’s folding or activity.

On the other hand, oxidative agents like iodine, hydrogen peroxide, and copper ion can be used to increase the oxidation state of cysteine residues, promoting disulfide bond formation. Changes in the state of the disulfide bonds could lead to new function or destabilize protein conformation.

2. Breaking Disulfide Bonds by Heat

Exposure to heat is another way to break disulfide bonds. When heated, proteins undergo denaturation, which is the break apart of the folded structure of the protein.

During this process, the protein structure unfolds, and disulfide bond-thiol interactions are disrupted. Disulfide-thiol exchange reactions also occur, with the thiol (-SH) group of a cysteine residue attacking the disulfide bond and liberating the protein from its structure.

In general, proteins that have a higher number of disulfide bonds and those with stronger bonds are harder to denature. Recently, metal-thiolate switch (MTS) technology was introduced as a way of tuning proteins activity using heat without resorting to heat denaturation. MTS technique exploits the bond between copper ion and cysteine residues. Copper ions will form a coordinative bond with a cysteine sulfur. This bond is stronger than normal disulfide bond. Heating the protein at high temperatures would break disulfide bonds but not break copper-cysteine bond because the bond energy is higher.

In this way, the protein is more stable even if it is subjected to heat.

3. Breaking Disulfide Bonds by Water

Water can also indirectly break disulfide bonds in alkaline hydrolysis. Alkaline hydrolysis is a chemical reaction that occurs when an alkaline solution (pH > 7) is applied to proteins rich in cysteine residues, where OH- ions attack either the polarized carbon atom adjacent to the thiol group (-CO-SH) or the bond linking the two cysteine residues in a disulfide bond. This reaction results in the formation of a thiolate anion (-C(SH)O-) and the release of the free cysteine residues.

Disulfide Bond and Protein Stability

The equilibrium between the folded and unfolded states of a protein is a function of several parameters, including entropy, enthalpy, and free energy. Disulfide bonds play a crucial role in maintaining the stability of the folded state.

Proteins with disulfide bonds are generally more stable than those with only intramolecular hydrogen bonds. The stability of a protein’s folded state is explained by the lowering of its free energy. The more extensive the disulfide-bonded network, the more energy is required to break it.

One way to determine the stability of a protein with disulfide bonds is through the measurement of its melting temperature (Tm). The Tm is the temperature at which 50% of the protein is unfolded when subjected to increasing heat. The Tm is affected by several factors, including the number of disulfide bonds present, the strength of the bonds, and the nature of the amino acid residues surrounding the disulfide bond.

Disrupting or removing disulfide bonds through reducing agents or other methods can reduce protein stability and cause their denaturation.


Disulfide bonds are essential for maintaining protein stability and structure. Breaking disulfide bonds can be achieved through various means, including oxidation, heat, and water. Researchers use these methods to study protein folding, activity, and therapeutics. Nonetheless, understanding the effect of disulfide bonds on protein stability can help guide the optimization of engineered proteins or even in drug discovery efforts. In summary, disulfide bonds are covalent bonds critical to maintaining the three-dimensional structure of proteins and aid in functions including stabilization, redox processes, protein folding, and biological processes. Different methods can break disulfide bonds, such as oxidation, heat, and water. Disrupting or removing disulfide bonds can significantly impact protein stability and structure.

The knowledge of disulfide bonds’ properties and functions can guide optimizing engineered proteins and drug discovery efforts.


  1. What are disulfide bonds in proteins?

    Disulfide bonds are strong covalent bonds between two cysteine residues that help proteins maintain their three-dimensional structures.

  2. How can disulfide bonds be broken?

    Disulfide bonds can be broken through oxidation, heat, or water exposure.

  3. What is the importance of disulfide bonds in protein stability?

    Disulfide bonds play a crucial role in maintaining protein stability and structure, affecting equilibrium, folding, and melting temperature, and therefore impact protein functions.

  4. Can protein stability be affected by the presence of disulfide bonds?

    Yes, protein stability is affected by several factors, including the number of disulfide bonds present in the protein and their strength.

  5. What are common reducing agents used to break disulfide bonds?

    Common reducing agents used to break disulfide bonds include -mercaptoethanol and dithiothritol (DTT).

  6. Why are disulfide bonds important in drug discovery?

    Understanding the effect of disulfide bonds on protein stability and function can guide the optimization of engineered proteins and drug discovery efforts.

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