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

Spatial Organization of the Protein Molecule
Methods for Studying the Secondary Structure of Proteins and Polypeptides
Deuterium Exchange

Another quantitative method for measuring the number of peptide groups interconnected by Hydrogen Bonds is The Study of The kinetics of isotopic exchange between the imide hydrogen of a protein or polypeptide and Water hydrogen. Although this technique has been briefly outlined above, it warrants a more detailed examination. The Essence of this method is as follows.

When a protein or polypeptide is dissolved in heavy water (D2O), its hydrogen atoms are replaced by D atoms at a rate that depends on the specific atoms to which they are bonded. It is well established that H^- atoms bonded to oxygen and nitrogen—including those in peptide groups—exchange very rapidly, whereas carbon-bonded atoms undergo slow exchange only at elevated temperatures. Based on this principle, it is theoretically possible to calculate the expected number of rapidly exchanging hydrogen atoms for a given polypeptide or protein.

On the other hand, it is known that in low-molecular-weight Polypeptides, the imide hydrogen of the peptide group exchanges with water immeasurably fast, whereas in high-molecular-weight polypeptides, this exchange is retarded and Temperature-dependent. The transition from rapid to slow exchange of the imide hydrogen with the medium serves as an indicator of intramolecular Hydrogen bond formation. By knowing the total number of rapidly exchanging imide hydrogen atoms and determining the quantity of slowly exchanging ones, we can ascertain the number of intramolecular Hydrogen bonds and, consequently, the degree of protein helicity.

However, the data obtained by this method are rather relative, as the clear-cut differentiation between rapidly and slowly exchanging hydrogen atoms is often difficult. The reason for this is as follows. It is known that The formation of various secondary bonds (such as disulfide bridges or side-chain interactions) can induce localized stress within the a-helix, leading to weakened hydrogen bonds. As a consequence, the imide hydrogen atoms in these units will exchange somewhat faster than those in other peptide groups, yet slower than in the complete absence of hydrogen bonds. For instance, out of 123 peptide hydrogens in Ribonuclease, 70 exchange slowly at 0°, meaning the degree of helicity can be estimated at 57%. However, among these 70 H-bonded imide hydrogens, 25 are capable of exchanging with water at 0° over the course of several days, another 25 exchange at 38° within a day, and only 20 resist exchange up to the melting temperature of the a-helix. Consequently, the degree of helicity can be estimated at either 36% (45 slowly exchanging H-atoms) or 16% (20 non-exchanging H-atoms). Therefore, the deuteration rate method can be used to assess protein helicity only in conjunction with other analytical techniques.

In Conclusion, two further Methods for evaluating the Introduction/11.html">Secondary Structure of Polypeptides and certain Proteins deserve mention: Electron Cell/15.html">Microscopy and Infrared Spectroscopy. These methods provide direct Evidence for the existence of a-helices and clearly identify the type of macromolecular secondary structure, respectively. However, these investigative techniques differ fundamentally from those discussed above in that they cannot be applied to study proteins and polypeptides in solution.