Protein Chemistry - Part 1 - General Protein Chemistry - Ashmarin, I. P. 1968

Protein Denaturation
Denaturing Agents

Denaturing agents are conventionally classified into chemical, physical, and biological. Although this Classification is convenient, it is somewhat arbitrary, as the Denaturation process is usually triggered by several interrelated factors (for example, Temperature and hydrogen ion concentration).

Chemical Agents. Many Proteins are denatured at extreme hydrogen ion concentrations—typically at a pH below 2–3 or above 10–11. Under these conditions, denaturation is apparently caused by the fact that virtually all polar groups carry like charges (predominantly H₃N groups at low pH and COO^- groups at high pH). This leads to mutual electrostatic repulsion among these groups and a disruption of the unique arrangement of polypeptide chains, accompanied by the Cleavage of some weak bonds that maintain the native Structure. This MECHANISM OF ACTION of extreme hydrogen ion concentrations is well illustrated by Experiments on the "melting" of a-Helical structures in synthetic Polypeptides composed of either dicarboxylic Amino Acids or Diaminomonocarboxylic Acids in the alkaline and acidic pH ranges, respectively.

It should be noted that A number of proteins are quite stable at extreme pH values. For instance, Histones, protamines, Trypsin, Chymotrypsin, Ribonuclease, Lysozyme, and several other proteins remain fairly stable at pH 2. Lysozyme, histones, and protamines are also stable at pH 10. Finally, there are proteins that undergo denaturation even within their "stability zone." For example, Myosin is denatured at a pH as early as 6.0, hemocyanin and Lipoproteins at ~pH 5, and Pepsin at pH 6.

Denaturation can be induced by a number of organic substances and Solvents. Chief among these organic substances are urea, guanidine salts, urethane, and similar amides. Their action is associated with the presence of the amide group, which enables them to "compete" with the protein's peptide groups for hydrogen bonding, redirecting these bonds to themselves. This leads to the dissociation of certain proteins into individual subunits or the transition of polypeptide chains from a helical form to a random coil. To exert their maximal effect, amides must be present in high concentrations—for instance, reaching up to 8 M in the case of urea.

The next group of denaturing organic substances—ionic detergents (such as sodium dodecyl sulfate) and sodium salicylate—apparently operates via a different mechanism. This mechanism involves the stoichiometric binding of the denaturing agents to oppositely charged protein groups. The electrostatic repulsion between like-charged groups remaining within the chain results in the cleavage of hydrogen and other bonds that stabilize the Native State.

Organic substances such as trichloroacetic acid and so-called alkaloid precipitants (picric acid, tannin, etc.) feature a complex denaturing mechanism that combines a direct impact on the hydrogen bonding system with the blocking of polar groups.

Interestingly, Protein Denaturation caused by all the aforementioned organic substances is temperature-independent and, in the case of urea, may even exhibit a negative temperature coefficient. This substantially differentiates them from organic solvents such as alcohol or acetone. The latter denature and irreversibly precipitate proteins from solutions at room temperature and above. At a critical temperature around 65°, protein precipitation occurs at relatively low concentrations of organic solvents. Nevertheless, as previously mentioned in Chapter I, protein fractionation can be performed near the freezing point by adding varying amounts of solvents without causing denaturation. It is believed that the precipitating and denaturing

action of organic solvents is related to their effect on the Dielectric Constant of the solution. The dielectric constant decreases significantly, which enhances the electrostatic interaction forces between charged groups both within the molecule and between different molecules. The formation of robust intra- and intermolecular salt-like bonds ultimately leads to irreversible protein coagulation.

Finally, among chemical agents, mention must be made of such denaturants as heavy Metal Ions, as well as thiocyanate and iodide ions. These substances apparently form fairly stable compounds with the polar groups of proteins, disrupting the ionic and hydrogen bonding System of the native protein molecule.

Physical Agents. Protein denaturation can also be brought about by various physical agents. The most common and thoroughly studied denaturing Treatment is heating. The thermal motion of polypeptide chains induces both the cleavage of Hydrogen Bonds between them and the disruption of hydrophobic group interactions. With a gradual increase in temperature, one can sometimes observe signs of a stepwise, abrupt course of denaturation. Apparently, The process of Hydrogen bond destruction in native molecules is cooperative in nature, allowing us to speak of the melting temperature and heat of fusion of a-helical segments in a number of proteins. Heat-denatured proteins readily aggregate and precipitate, although coagulation is a secondary phenomenon. Coagulation is likely the result of the formation of additional disulfide bridges, salt-like bonds, and secondary hydrogen bonds between different molecules. The fact that coagulation is closely linked to disulfide bond formation is supported by the observation that p-chloromercuribenzoate inhibits aggregation. Conversely, Collagen, which lacks sulfhydryl groups, converts into soluble gelatin upon heating.

Since aggregation invariably accompanies thermal denaturation, The amount of precipitated protein can serve as an indicator of the process rate. Furthermore, The rate of thermal denaturation is highly dependent on the pH of the medium: it reaches a minimum at the isoelectric point and increases when the pH shifts in either direction away from it.

It should be noted that alongside the vast majority of thermolabile proteins, there are proteins that are remarkably resistant to heat. These primarily include proteins from Bacteria thriving in hot springs, as well as ribonuclease, lysozyme, trypsin, chymotrypsin, and several others.

Proteins undergo denaturation under mechanical stresses such as high pressure (around 5,000–10,000 atm), grinding of dry preparations, vigorous shaking of solutions, and spreading to form a surface film. Denaturation upon spreading is explained by the formation of a protein monolayer at the interface, within which polypeptide chains unfold. Protein sensitivity to these treatments varies considerably: egg albumin, for instance, is very easily denatured by shaking its solution, whereas The activity of Insulin remains unaffected. Lipoproteins are prone to denaturation and dissociation upon freezing. Mechanical denaturing treatments also include irradiation with high-frequency sound waves. This must be taken into account when using ultrasonic generators for Cell Disruption to extract proteins. At a frequency of about 10 kHz and with careful cooling of the sonicated solution, denaturation is usually negligible or entirely absent.

Proteins are denatured upon exposure to ultraviolet light, as well as high doses of X-rays and y-rays. The Effect of ultraviolet light is particularly pronounced at wavelengths from 260 to 310 mµ, with primary absorption attributed to Tyrosine and Tryptophan rings. It has been demonstrated that prolonged exposure to ultraviolet rays results in the cleavage of peptide bonds adjacent to aromatic rings. Ionizing radiation can likewise cause breaks between a-carbon atoms and amino acid side chains. In addition, secondary processes may occur due to the generation of various free radicals such as O₂H, H₂O₂, OH, etc. The latter are strong oxidizing agents that attack thiol groups and cleave Disulfide Bonds.

Biological Agents. Finally, protein denaturation apparently also occurs under the action of Proteolytic Enzymes. It is well known that the latter readily digest only denatured proteins. This suggests that the initial stage of these enzymes' action on a protein consists of the denaturation of its molecules or a structural alteration that exposes the most crucial peptide bonds to cleavage. An indicator of denaturation during the initial stage of proteolysis is an increase in negative optical rotation and a decrease in solution volume, observed to the same extent as during urea-induced protein denaturation.

In concluding this Brief Overview of agents causing protein denaturation, mention must be made of Methods to prevent this process. Some of these were already discussed at the beginning of Chapter I in connection with the purification and storage of protein preparations. Therefore, we shall limit ourselves to recalling the stabilizing effect of low temperatures, high concentrations of certain neutral salts, and the protein itself, The Importance of solution pH, and, finally, the stabilization of protein preparations via vacuum freeze-drying (lyophilization). In addition, a number of other factors are known to protect proteins from denaturation. For example, the thermal denaturation of several proteins is inhibited by concentrated solutions of sugars (glucose, fructose, sucrose, etc.) and polyhydric alcohols. Effective protection against the denaturing action of urea, guanidine hydrochloride, and heat is provided by low concentrations of long-chain organic ions, fatty acid anions (sodium caprylate), and detergents (sodium dodecyl sulfate). Interestingly, low concentrations of such denaturing agents as urea can once again protect certain proteins (e.g., serum albumin) against thermal denaturation. The action of these agents is likely due to their binding by protein particles to form hydrophilic complexes. In such complexes, the protein is coated with a shell of adsorbed molecules that hinders the formation of aggregates with other protein particles.