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
Infrared Spectroscopy

It is well known that the infrared region of the spectrum generally refers to the range extending from the edge of visible light (approximately 7500 Å, or 0.75 µm) to the lower limit of the microwave region (approximately 1000 µm). Infrared radiation is typically characterized by frequency rather than wavelength. The universally accepted unit of frequency is the wave number (cm^-1), which expresses the number of wavelengths per centimeter. Of primary interest to biochemistry is a relatively narrow frequency interval from 4000 to 625 cm^-1 (2.5–16 µm), since it is within these limits that the absorption bands associated with the molecular Structure of Proteins and Polypeptides are located.

The infrared absorption spectrum of these compounds consists of 20–30 discrete absorption bands, of which 5–10 are more intense than the rest. The presence of absorption bands in the spectrum is due to the fact that the atoms of a protein molecule oscillate (displace) at a specific frequency along or perpendicular to the direction of their chemical bonds, causing periodic Changes in the dipole moment of the molecule. As a result, a polyatomic molecule can absorb radiation of the corresponding frequencies. The high intensity of certain bands is explained by the fact that the changes in dipole moment corresponding to certain frequencies are the largest. Although all atoms of the molecule participate to some extent in each fundamental vibration, many of the characteristic frequencies are primarily due to the vibrations of specific chemical bonds or individual groups comprising the molecule. These frequencies can be used to establish the presence of corresponding groups in polyatomic molecules (group frequencies). Of greatest interest for the analysis of PROTEINS AND Peptides are the stretching frequencies of hydrogen atoms, hydrogen deformation frequencies, multiple bond frequencies, and backbone frequencies.

The hydrogen stretching frequencies correspond to the stretching and contraction of C—H, N—H, O—H, S—H, and P—H bonds and characterize the motion of H-atoms along these bonds. The most important among them are the following:

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Under METABOLISM/18.html">The Influence of Hydrogen Bonds, the last two frequencies are shifted downward by several hundred cm^-1, and the absorption bands become broadened.

Hydrogen deformation frequencies are associated with the motion of hydrogen atoms nearly perpendicular to the chemical bonds and lie between 625 and 1670 cm^-1 (1.6–6 µm). Under the influence of hydrogen bonds, these frequencies increase slightly; the absorption bands widen insignificantly.

Multiple bond frequencies arise from changes in the distances between atoms along double and triple bonds. Of particular interest are the frequencies of the carbonyl group C—O, which are observed between 1670 and 1820 cm^-1 and partially overlap with the hydrogen deformation frequencies. Therefore, it is sometimes difficult to distinguish between the stretching vibrations of a double bond and the deformation vibration of a hydrogen atom. The Effect of hydrogen bonds lowers these frequencies insignificantly.

Finally, the vibrational frequencies of the main backbone of valence bonds (skeletal frequencies) are caused by the expansion and contraction of single bonds such as C—C, C—O, and C—N. These frequencies are located in the range of 200–1250 cm^-1. Unfortunately, they cannot be used to determine The structure of polypeptides and proteins due to the lack of data on THE ORIGIN OF each.

By having the absorption spectrum of a protein or polypeptide and knowing the group frequencies, one can establish only the presence of the corresponding groups in their molecule. By measuring the optical density at these frequencies, one could, with appropriate calibration, perform a Quantitative determination of these groups. However, the structure of proteins has not yet been studied sufficiently, which complicates precise analysis. Therefore, the main research technique is the quantitative measurement of polarized infrared Light absorption by oriented polymer samples. These measurements make it possible to determine the spatial arrangement of chemical bonds in protein and polypeptide molecules.

THE PRINCIPLE OF this method is as follows. Thin films are prepared from oriented polymers by rolling or casting, in which the polymer molecules are partially ordered relative to a certain axis. Then, the absorption of infrared light polarized parallel and perpendicular to the orientation axis is measured. The measurement results are expressed as the difference in absorption for each maximum. This difference in the absorption of light polarized parallel and perpendicular to the molecular orientation axis is called the dichroic ratio. From the magnitude of the dichroic ratio, the direction of chemical bond vibrations relative to the orientation direction can be determined.

Fig. 23. Spectra of synthetic polypeptides in polarized infrared light (from Neurath and Bailey, 1956).

Solid line — light vector perpendicular to the orientation direction; dashed line — light vector parallel to the orientation direction; a — poly-y-benzoyl-L-glutamate from a solution in chloroform (a-form); b — copolymer of poly-y-methyl-L-glutamate-D,L-phenylalanine from a solution in formic acid (B-form).

To illustrate the analysis principle discussed above, let us examine the spectra of synthetic polypeptides in polarized infrared light (Fig. 23). Films of poly-y-benzoyl-L-glutamate from a chloroform solution (a-form) and a copolymer of poly-y-methyl-L-glutamate D,L-phenylalanine from a formic acid solution (ß-form) were taken as oriented samples. Both polypeptides exhibit the following main absorption bands: 3300 and 3080 cm^-1; 1750 cm^-1, 1650 cm^-1, and 1550 cm^-1. The first two and the fourth absorption bands correspond to the stretching vibrations of N—H and C=O groups, modified by the action of hydrogen bonds. To verify the arrangement of these bonds (intra-chain or inter-chain), the absorption of light polarized parallel and perpendicular to the orientation direction is measured. It can be seen that for the a-form of the polypeptide, light absorption is significantly greater when the light is polarized along the orientation axis (parallel dichroism). For the ß-form, we observe the reverse dichroism. Consequently, in the a-form, the direction of vibration of the N—H and C=O groups is parallel to the orientation axis (intramolecular H-bonds), whereas in the ß-form it is perpendicular to it, and hydrogen bonds link individual polypeptide chains together.

Thus, by determining the dichroic ratio, one can establish the orientation of the most important groups of the protein macromolecule (N—H and C—O) relative to its axis and thereby determine its Secondary structure.