Practical Protein Chemistry - A. Darbre 1989

Determination of C-Terminal Amino Acid Sequence
Isolation and Identification of C-Terminal Peptides
Two-Dimensional Peptide Mapping

To date, several Methods of peptide mapping have been proposed. The C-terminal peptide of the fetal Hemoglobin y-chain was identified by comparing the electrophoretograms of tryptic hydrolysates following secondary Separation (pH 6.5) in a direction perpendicular to the first, both before and after the removal of C-terminal Arg or Lys residues using carboxypeptidase B directly on the filter paper [63]. Theoretically, following carboxypeptidase Treatment, only the C-terminal peptide should lie along the diagonal. However, in the case of human hemoglobin 7-chain, 3 spots were found on the diagonal, namely free Lys, the dipeptide Val-Lys (since dipeptides are cleaved very slowly by carboxypeptidase [46]), and the expected C-terminal peptide Tyr-His. This method is suitable for Proteins whose C-terminal amino acid is any residue other than Arg and Lys, as well as for those proteins whose tryptic Peptides are readily cleaved by carboxypeptidase B.

To detect the C-terminal region of bacterial Tryptophan synthetase A-protein, peptide maps of the protein digest before and after treatment with a mixture of Carboxypeptidases A and B were compared [15]. The C-terminal sequence of this protein has the composition Ala-Ala-Thr-Arg-Ser, and upon partial tryptic Hydrolysis, three spots corresponding to Ala-Ala-Thr-Arg-Ser, Ala-Ala-Thr-Arg, and free Ser are revealed on the peptide map. Following carboxypeptidase treatment, only the Ala-Ala-Thr-Arg spot disappears because the second peptide and free Ser migrate on the chromatogram, overlapping with other spots. The successful application of this method strongly depends on the completeness of peptide separation on the chromatogram. The larger the protein molecule, the higher the probability that the C-terminal peptide will be lost.

Among all peptide mapping methods employed, the most elegant is the diagonal Electrophoresis (EP) method, which relies on the absence of a free carboxyl group in amidated proteins. Prior to enzymatic Cleavage, the carboxyl groups of the side chains and the C-terminal amino acid are amidated. Following hydrolysis, the C-terminal fragment turns out to be the only one lacking a free carboxyl group. It can be selectively identified because its ionic charge, and consequently its electrophoretic mobility, remain virtually unchanged upon pH variation in either direction from pH 3.5 (the pK of the a-carboxyl group). The mobilities of peptides containing a free carboxyl group will increase upon acidification of the solution (pH<3.5). Following two-dimensional EP, only the C-terminal peptide is detected on the diagonal of the electrophoretogram.

In the original Procedure [27], the protein was amidated with methylamine using N-cyclohexyl-N'-2-(4-β-morpholinyl)ethylcarbodiimide p-toluenesulfonate; enzymatic cleavage was then carried out, and the hydrolysate was subjected to high-voltage EP at pH 6.5 in the first dimension and pH 1.8 In the second. Under these conditions, C-terminal peptides containing His are not detected as they deviate from the diagonal. In the modified procedure, this issue is resolved by performing the EP in the first dimension at pH 4.4; therefore, when conducting the separation in the perpendicular direction at pH 1.8, the carboxyl groups are the only ones that have altered their charge [22]. Ethanolamine was used for protein amidation because it is readily Water-soluble, improves the solubility of the modified protein and the resulting peptides, is absent from common proteins, and can be quantified on an amino acid analyzer. In [22], to determine the exact positions of substances located on the diagonal independently of minor shifts in absolute mobility or electroosmotic effects, Ala amide or taurine was used as markers.

18.2.3.1. Isolation of C-terminal peptides. The procedure for isolating the carboxyl-containing terminal peptides of human hemoglobin a- and ß-chains is described below [22]. The amount of protein required for the analysis depends on the sensitivity of the detection method.

Amidation. The protein (1.6 µmol, 50 mg) is dissolved in 3 mL of water containing a few drops of Hydrochloric acid. Then 2.6 g of urea is added and stirred for 30 min. Next, 0.3 g of potassium chloride and 0.2 mL of ethanolamine are added, and the pH is immediately adjusted to 4.75 with hydrochloric acid. EDC (125 mg) is added, and the pH is maintained at 4.75. The mixture is stirred at room Temperature for 4–6 h, after which 1 mL of 4 M sodium acetate (pH 4.75) is added. The solution is dialyzed against several changes of water.

Alternatively, amidation can be carried out in a saturated guanidine hydrochloride solution (1 mL) containing ethanolamine (20 µL, 300 µmol) at pH 4.7 (adjusted with 1 M hydrochloric acid using a microsyringe) [21]. Carbodiimide (EDC, 15 mg, 80 µmol) is added with stirring, and the pH is maintained at 4.7 for 6 h.

Enzymatic cleavage. Thermolytic cleavage is carried out in a 1% aqueous ammonium bicarbonate solution at 37 °C for 8 h at an Enzyme-to-substrate ratio of 1 : 35 (by weight). To stop the reaction, the mixture is lyophilized.

Isolation of peptides by electrophoresis. The peptide mixture is dissolved in pyridine – acetic acid – water buffer (6:10:1200, pH 4.4), applied as a 330 mm strip to Whatman 3MM Chromatography paper along with markers (Ala amide and taurine, 5 nmol/mm), and subjected to electrophoresis at pH 4.4 for 60 min at a voltage of 6 V/mm; the paper is then dried. For the analytical procedure, the strip containing the peptides is excised, stitched onto a whole sheet of Whatman 3MM paper, and subjected to electrophoresis at pH 2.1 (formic acid – acetic acid – water, 1:4:45) for 40 min at 6 V/mm. The electrophoretogram is stained with a Cd-ninhydrin solution (sec. 8.14.2). The electrophoretogram in Fig. 18.2 shows that two peptides lie on the diagonal bounded by the markers. (In a preparative procedure, following electrophoresis at pH 4.4, the region on the diagonal containing the two peptides can be excised and subjected to electrophoresis at pH 2.1.) Peptides migrating slower than the others and corresponding to the two "diagonal" peptides are excised and, if necessary, further purified by electrophoresis at pH 2.1 or 6.5.

This technique has been recently used to determine the cleavage site of the Influenza virus hemagglutinin molecule by the proteolytic enzyme bromelain during the enzymatic release of the protein from the viral surface [21].

This method is expected to become widely adopted [22]. All peptides derived from the C-terminal region of the protein lie on the diagonal regardless of their Amino Acid Sequence. Any enzyme can be used for hydrolysis, provided that the hydrolyzate contains peptides of sufficient length, and The sequence of the isolated peptide can be determined using established (standard) methods.

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FIG. 18.2. Selective isolation of C-terminal peptides from the thermolytic hydrolyzate of human hemoglobin $\alpha$- and $\beta$-chains. The hydrolyzate was separated by electrophoresis at pH 4.4 in the first dimension and at pH 2.1 in the second [21].

The presence of ethanolamine at the carboxyl terminus of the isolated peptide confirms that this peptide belongs to the C-terminus of the protein. Potential low yields of ethanolamine attachment to the protein are not a serious limitation of the method, since any non-amidated peptide is shifted off the diagonal after electrophoresis and therefore does not interfere with the identification of the target fragment. Finally, the method should be applicable to proteins with a naturally amidated C-terminal amino acid, as such proteins contain a blocking group of the same type as that produced by artificial amidation.