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

Primary structure of the protein molecule
Study of the sequence of amino acid residues in peptide chains

Once the individual polypeptide chains have been isolated, one can proceed to determine the Amino Acid Sequence. To this end, the chain must first be cleaved into a series of fragments, which can be achieved through incomplete Hydrolysis using 12 N Hydrochloric acid or Proteolytic Enzymes. In the former case, depending on the hydrolysis duration, A large number of di-, tri-, tetra-, and pentapeptides are obtained, many of which partially overlap with one another. Deciphering the residue sequence in short (di- and tri-) Peptides is relatively straightforward by identifying the terminal groups. However, when analyzing The Structure of tetra- and pentapeptides, this approach proves insufficient and must be combined with partial hydrolysis of the peptides themselves.

Let us decipher The sequence of a hypothetical pentapeptide (A, B, C, D, E), where the letters denote individual Amino Acids. First, the N-terminal amino acid must be identified—let us say it is D. The sequence can then be expressed as D—(A, B, C, E). Following partial hydrolysis of the D—(A, B, C, E) peptide, the dipeptides D—A, A—C, B—E, C—B, and two tripeptides D—(A, C) and A—(B, C) were isolated and analyzed for Amino Acid Composition and DNP-amino acids. The presence of the D—A dipeptide in the hydrolysis products reveals Two amino acids at the N-terminus of the pentapeptide in the D—A sequence. The identification of the A—C dipeptide and the A—(B, C) tripeptide extends the N-terminal sequence to D—A—C, and the A—(B, C) tripeptide can be decoded as A—C—B. The Isolation of the C—B dipeptide indicates B as the next adjacent residue in the D—A—C—B sequence. Only amino acid E remains, which is evidently the C-terminal amino acid. Thus, the complete sequence is expressed as D—A—C—B—E.

In the example given above, each amino acid occurs only once, which is extremely rare in practice. If an amino acid appears multiple times within a peptide, the arrangement of smaller peptides along the chain cannot be predicted with certainty, as this leads to additional overlapping regions that may belong to completely different sections of the polypeptide. For instance, if the hydrolysate contains two tripeptides, A—B—B and B—B—D, and three dipeptides, A—B, B—B, and B—D, it is quite plausible to assume the existence of a tripeptide A—B—D in the original chain, alongside the tetrapeptide A—B—B—D. On the other hand, the presence of a uniquely occurring amino acid residue often serves as a kind of focal point from which the arrangement of neighboring amino acids along the polypeptide chain can be readily extended.

Decoding The amino acid sequence in peptides obtained via incomplete acid hydrolysis and comparing them can sometimes help reconstruct larger fragments. However, more frequently, these data serve merely as an auxiliary tool for analyzing relatively large segments produced by the Enzymatic Cleavage of a polypeptide chain. This cleavage is performed using proteinases such as Trypsin, Chymotrypsin, Pepsin, and subtilisin. As mentioned earlier (Chapter II), trypsin is the most convenient among them due to its strict Specificity, cleaving peptide bonds exclusively at the carboxyl groups of Lysine and Arginine. Since trypsin is an endopeptidase, it breaks bonds only within the interior of the chain and does not release free amino acids. Chymotrypsin is less specific, cleaving peptide bonds at the carboxyl groups of Tyrosine, phenylalanine, Tryptophan, and Methionine. The specificities of pepsin and subtilisin are relatively broad, and they attack a variety of bonds (see Fig. 7).

The action of any single enzyme yields peptides that no longer overlap with one another, with their length depending on the positions of the amino acids cleaved by that enzyme within the original chain. Consequently, we obtain a mixture consisting of both short and relatively long peptides. To study the structure of these peptides, each must be isolated in pure form. ¹

The Separation of a large number of peptides generally cannot be achieved using a single technique; obtaining them in purified form requires combining several independent Methods. The most commonly used ones are briefly outlined below. Paper Chromatography, for instance, is the simplest, yet the most time-consuming and not always "cleanest" method. The initial mixture is separated using a single solvent into several groups, each containing several different peptides. Each group is then eluted from the paper and re-separated in a different solvent system. The resulting fractions are chromatographed again, each in a new solvent system, until homogeneous peptides are obtained. This separation process is very lengthy and requires a significant amount of starting material due to inevitable losses during chromatography and subsequent elutions.

A faster method for separating peptides from an enzymatic hydrolysate is peptide mapping (or "fingerprinting"), which combines high-voltage Electrophoresis and paper chromatography. This method makes it possible to compare peptide mixtures obtained from the Digestion of various Proteins and to detect even minor differences in the amino acid composition of individual peptides, such as the substitution of a single amino acid by another. Such differences are of great importance in the comparative study of Homologous proteins from various PLANT AND ANIMAL species, as well as in identifying genetic alterations in Cell/13.html">Protein Structure resulting from point Mutations.

This type of analysis consists of the parallel enzymatic digestion of the proteins being compared and the subsequent comparison of their electrochromatograms. To do this, a sample of the hydrolysate is applied to the corner of a large sheet of Whatman No. 3 chromatographic paper and subjected to electrophoresis at a voltage of approximately 1.5–3 kV in pyridine-acetate or acetate-formate Buffer solutions. During this process, the initial mixture is separated into a series of fractions based on differences in the electrochemical Properties of the respective protein fragments. However, electrophoresis alone cannot achieve complete separation of the peptide fragments. Therefore, upon completion of electrophoresis, the sheet is dried, and the spots are subjected to chromatographic separation in the perpendicular direction using various solvent systems. After chromatography, the sheet is dried and sprayed with a ninhydrin solution; the peptide spots develop upon brief heating at 70°C or by keeping the sheet in the dark for 12–24 hours. Provided that all conditions remain constant and standard Materials are used, the peptide maps are reproducible, and the positions of the spots from one experiment to another coincide with an accuracy of 3 to 5 mm.

If one of the proteins being compared exhibits differences in the amino acid sequence of a specific region of the primary chain, the peptide fragments from this region will migrate across the electrochromatographic field at different rates (and often in a different direction) compared to the fragments from the corresponding region of the other protein. As a result, the former peptides will occupy distinct positions on the peptide maps, whereas fragments from identical regions of The polypeptide chains will occupy corresponding places. Sequence differences will thus be detected as spots present on one electrochromatogram and absent on the other. Elution and quantitative Amino acid analysis of these "extra" peptide spots subsequently make it possible to establish the presence of protein-specific amino acids characteristic of these fragments. In particular, the peptide mapping method has been successfully used to reveal Structural Features of various Hemoglobins in tryptic hydrolysates. Hemoglobin S differs from normal hemoglobin A in its electrophoretic mobility and partially replaces the latter in patients with Sickle-Cell Anemia. Peptide mapping studies have localized the Primary Structure difference between hemoglobins A and S to a single site on the polypeptide chain:

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When comparing the peptide maps of the tryptic hydrolysate, peptide 4 of hemoglobin S is seen to segregate from peptides 1, 2, and 3, which is due to the substitution of valine for glutamic acid in its composition (Fig. 10).

Gel filtration is occasionally employed to separate peptides from an enzymatic hydrolysate. THE PRINCIPLE OF this method has been outlined above (Chapter I); therefore, it suffices to note that Sephadex G-25 is the most suitable medium for this purpose.

Fig. 10. Peptide distribution maps of tryptic hydrolysates of hemoglobins A and S (from Bailey, 1965).

Dashed lines indicate peptides that appear only after heating the chromatogram.

A wide variety of solutions are used to equilibrate the Column and elute the peptides, including Water, sodium chloride solutions, ammonia, acetic acid, and various buffer solutions. This method has been used to fractionate peptide hydrolysates of human hemoglobin, alpha-casein, Skeletal Muscle Tropomyosin, and trypsin autolysate.

It should be noted, however, that fractions eluted from Sephadex are generally not homogeneous and contain multiple peptides (up to 10 or more). Consequently, this separation method must be combined with fractionation techniques such as fingerprinting and Ion-exchange chromatography.

Ion-exchange chromatography is the most sophisticated technique for peptide separation. As with amino acid chromatography, polystyrene resins such as Dowex are used for this purpose, with the choice of resin (anion or cation exchanger) depending on The Nature of the peptides to be separated. Chromatography of acidic and neutral peptides is best performed on strong anion exchangers (Dowex-1, with the active ionogenic group being a quaternary ammonium base), whereas strong cation exchangers (Dowex-50, with the active ionogenic group being SO₃H) are preferred for neutral and basic peptides. Frequently, to determine the appropriate resin type, the peptide mixture is separated electrophoretically to ascertain whether acidic or basic peptides predominate. Thus, the Selection of the resin is largely empirical.

The fractionation of peptides on ion-exchange resins is based on A number of principles discussed earlier (Chapters I and II). These include differences in the electrochemical properties of the separated fragments, varying affinities of nonpolar radicals for the benzene rings of the resin matrix, and Changes in the concentration of competing ions in the buffer solution. Therefore, the elution of peptides from the resin is accomplished by passing buffer solutions of varying Ionic strength and pH through the column. This adjustment of ion concentration and pH can be performed linearly or stepwise. The eluted components are identified by ninhydrin colorimetry, lyophilized (freeze-dried), and tested for homogeneity using paper chromatography and peptide mapping. Non-homogeneous fractions are subjected to further fractionation. As an example, one can cite the separation of peptides obtained by the tryptic digestion of oxidized (i.e., disulfide-bond-free) Ribonuclease (Fig. 11).

Thus, following the cleavage of a polypeptide chain by an enzyme (such as trypsin) and the separation of the hydrolysis products using the methods listed above, a large number of homogeneous peptide fragments can be obtained. The amino acid sequence within these fragments must then be determined. For short peptides, this task is relatively straightforward and is accomplished by determining the amino acid composition and the sequence of a few amino acids from the N- or C-terminus of the fragment. If a peptide consists of a large number of residues, it must be further cleaved into smaller segments using a second proteolytic enzyme (e.g., chymotrypsin). By controlling the extent of digestion in each experiment through the adjustment of reaction time, pH, and Temperature, a series of peptides of varying sizes can be obtained, many of which will partially overlap. By comparing these fragments, the sequence of amino acid residues in the original large peptide can be reconstructed. A specific example of such an analysis is given below.

For instance, one of the peptide fragments from the tryptic hydrolysate of ribonuclease consisted of 20 amino acid residues and possessed Histidine and valine as its N- and C-terminal amino acids, respectively:

H—His—(Val₃, Ile₂, Cys, Asp-NH₂, Glu∙NH₂, Gly,

Ala₂, Pro₂, Tyr, His, Phe, Asp, Ser)—Val—OH

Fig. 11. Separation of peptides from the tryptic hydrolysate of oxidized ribonuclease on a Dowex 50-X2 cation-exchange column (from Harris and Ingram, 1963):

Column size 150 × 2 cm. Peaks 1, 2, 3, 4, 9, 13 + 14 are unidentified peptides, 5 is (Cys, Asn, Ala, Val)—Lys, 6 is (Glu, Gly, Ser₂, Thr)—Lys, 7 is Asn, Arg, 8 is (Asn, Gln, Ala, Thr₂)—Lys, 10 is Lys—Glu—Thr—Ala—Ala—Ala—Lys, 11 is (Asn, Leu, Thr)—Lys, 12 is Ser-Arg, 15 is (Gln, Phe)—Arg.

Controlled hydrolysis of this fragment with chymotrypsin and pepsin yielded a set of overlapping peptides (overlap regions are indicated by dashed lines). Comparison of these peptides made it possible to establish the amino acid sequence in the original fragment (Fig. 12).

Similar techniques were used to determine the sequence of all other peptides obtained by trypsin digestion of ribonuclease. However, this was not yet sufficient to establish the order of amino acid residues throughout the entire polypeptide chain of ribonuclease, as the exact Location of each peptide within the chain remained unclear.

To solve this problem, it was necessary to subject the entire polypeptide chain of ribonuclease to Enzymatic hydrolysis once again, but using a different enzyme. While trypsin was used initially, the subsequent cleavage was carried out with chymotrypsin. The peptides obtained from chymotryptic hydrolysis were isolated in pure form and analyzed for the sequence of amino acid residues. The goal of this monumental work was to obtain several sets of partially overlapping peptides. Having such sets of overlapping peptides makes it possible to determine not only the amino acid sequence within individual peptides, but also the points where these peptides overlap and join within the single polypeptide chain. In other words, this approach allows one to establish the order of residues in the primary chain of the whole protein. It should be noted that one often has to resort to hydrolysis of the chain using a third (pepsin) and sometimes even a fourth (Papain) enzyme. It was precisely this method that was used to decipher the Introduction/19.html">Primary structure of the A- and B-chains of Insulin (Figs. 13, 14), ribonuclease, cytochrome c, and other proteins.