Human Biochemistry, Volume 1 - Murray R. 1993

Structure and Functions of Proteins and Enzymes
Peptides
Automated Edman degradation for determining the amino acid sequence of polypeptides

Separation into Linear Polypeptides

Many protein molecules contain more than one polypeptide chain; these chains are linked either by noncovalent bonds or by disulfide bridges. Therefore, the first step must be the isolation of individual polypeptide chains. The dissociation of noncovalently linked polypeptides is carried out using Denaturing Agents (such as urea or guanidine hydrochloride) that disrupt Hydrogen Bonds. Disulfide bridges are cleaved using oxidizing and reducing agents (Fig. 4.10). Subsequently, chromatographic separation of the polypeptides is performed.

Cleavage of the Polypeptide into Fragments Suitable for Automated Sequencing

Automated sequenators operate most efficiently when the polypeptide length is between 20 and 60 residues. These requirements have largely driven The Development of Methods for cleaving polypeptides into fragments of the desired size and purifying them. The primary objective is not to obtain A large number of small fragments suitable for manual sequencing, but rather to cleave the protein into a small number of large fragments (from 30 to 100 residues). At the same time, it is desirable to achieve complete, highly Specific Cleavage at a limited number of bonds. Cyanogen bromide (CNBr), Trypsin, and o-iodosobenzoic acid meet these requirements.

A. CNBr. Cysteine residues are first modified with iodoacetic acid. Subsequently, CNBr specifically cleaves the peptide bonds on the COOH side of Methionine residues, in most cases with a quantitative yield. Methionine residues occur relatively infrequently in polypeptides, so such cleavage yields peptide fragments of the desired size.

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Fig. 4.10. Two adjacent polypeptide chains connected by a disulfide bond (shaded). Cleavage of this bond by oxidation with performic acid (left) or by reduction with ß-mercaptoethanol (right) yields two Peptides containing cysteic acid or cysteine residues, respectively.

B. Trypsin. Trypsin cleaves bonds on the COOH side of Lysine and Arginine residues. To limit the number of cleavage sites, lysine residues are pre-modified with citraconic anhydride (a reversible reaction); this converts the positive charge of the lysine residues into a negative charge. Modifying arginine residues is less practical since lysine residues occur relatively more frequently. However, it can be useful for the subsequent cleavage of CNBr fragments.

C. o-Iodosobenzoic acid. This reagent specifically and quantitatively cleaves relatively rare Trp-X bonds. Preliminary protection of other residues is not required.

D. Hydroxylamine. Hydroxylamine cleaves the relatively infrequent Asn-Gly bonds, although the yield is not quantitative.

E. Staphylococcus aureus V8 protease. This enzyme cleaves Glu-X bonds, predominantly when X is a hydrophobic residue. The Glu-Lys bond is not cleaved. This reaction is also useful for the subsequent cleavage of CNBr fragments.

F. Mild acid Hydrolysis. The infrequently occurring Asp-Pro bonds are susceptible to cleavage by this method.

Two to three sets of fragments—typically obtained by cleaving the original polypeptide at Met, Trp, Arg residues and at Asn-Gly bonds—along with subfragments obtained from secondary cleavage, are usually sufficient to determine the complete Introduction/19.html">Primary Structure of a polypeptide. Provided there are no unforeseen difficulties in fragment purification and with due care, the entire Procedure requires only a few micromoles of polypeptide.

Fragments are purified by Gel filtration in acetic or formic acid (Fig. 4.7), high-pressure reverse-phase liquid Chromatography (Fig. 4.8), or Ion-exchange chromatography on phosphocellulose or sulfopropyl-Sephadex.

Edman Reagent and the Edman Degradation

In automated sequencing, phenylisothiocyanate (the Edman reagent) is used instead of Sanger's reagent; the reaction results in the cleavage of the N-terminal residue as its phenylthiohydantoin derivative (the Edman degradation; Fig. 4.11).

All reactions take place within a liquid film coating the walls of a rotating cylindrical chamber, which facilitates extraction and subsequent solvent removal. Several manufacturers produce instruments capable of fully automated sequence analysis of polypeptides containing up to 30–40 residues (in some cases up to 60 or even 80 residues) per run. The instrument is programmed for the sequential Edman cleavage of the polypeptide's N-terminal residues. Following the cleavage, isolation, and identification of the original N-terminal amino acid (Fig. 4.11), the Edman derivative of the next amino acid is formed, and so on. Phenylthiohydantoin derivatives are identified using high-pressure liquid chromatography. This device allows for the determination of significantly longer sequences than manual sequencing, and does so much faster.

Fig. 4.11. Formation of phenylthiohydantoin from an amino acid (or the N-terminal residue of a polypeptide). Phenylisothiocyanate reacts with the amino groups of Amino Acids or peptides to yield phenylthiohydantoin derivatives. Subsequent Treatment with acid in Solvents lacking hydroxyl groups induces cyclization of these derivatives to form phenylthiohydantoins. This reaction, which allows the identification of the peptide's N-terminal residue, is utilized in the automated sequencing of polypeptides.

Determination of the Complete Primary Structure by Comparing Overlapping Peptide Sequences

The final step consists of reconstructing the original sequence in which the sequenced peptides were arranged within the native protein. To achieve this, one must have peptides generated by different methods that cleave the protein chain at various sites (e.g., using trypsin and Chymotrypsin). By aligning the sequences of these overlapping peptides, the primary structure is unambiguously established (Fig. 4.12).

Fig. 4.12. Using peptide Z, whose sequence overlaps with those of peptides X and Y, it can be established that in the original protein, peptides X and Y are arranged in the order X→Y rather than Y→X.