Practical Protein Chemistry - A. Darbre 1989

Determination of the C-terminal amino acid sequence
Determination of the C-terminal sequence
Cleavage by means of thiocyanate

There are currently no Methods available for determining long sequences of C-terminal Amino Acids. The chemical and enzymatic approaches currently in use allow The sequence of only a few residues to be determined. In most cases, Carboxypeptidases are primarily used to obtain this information, despite the inherent drawbacks of this method: varying Cleavage rates for different Amino Acids and the difficulty of maintaining the carefully controlled conditions required for stepwise cleavage. Issues related to The Use of carboxypeptidases are discussed in Section 18.4.4.

Among all chemical approaches, this method has been the most thoroughly investigated [83]. It was later modified to improve reaction conditions in solution [18, 48, 84, 97] and on solid Supports [71, 94]. However, each of the proposed variations has made it possible to determine the sequence of only a few C-terminal amino acids.

The peptide (protein) is incubated with ammonium thioacetate in a solution of acetic acid and acetic anhydride to form a peptidyl isothiocyanate, which spontaneously rearranges into a peptidyl thiohydantoin (Fig. 18.9). The latter is selectively cleaved using acid or alkali, yielding the thiohydantoin of the C-terminal Amino Acid and a shortened peptide (protein) with a new C-terminal residue.

This method for C-terminal Sequence Determination is based on the work of [41] concerning The conversion of acylamino acids into thiohydantoins using ammonium thiocyanate and acetic anhydride. Later [76], a cleavage step (1 M sodium hydroxide, 3 h, room Temperature) was added to this reaction, making it possible to transition to the repetitive formation and cleavage of amino acid thiohydantoins, i.e., to the Analysis of the C-terminal sequence of Polypeptides. The latter technique has been applied with limited success to determine the Terminal Groups of A number of Peptides and Proteins [29]. Revising the conditions for thiohydantoin formation and cleavage led to The Development of a Procedure for determining Amino acid sequences in peptides under milder conditions [83]. The C-terminal sequence of Proteins can also be determined using the same scheme [18]. However, under these specified conditions, Asp and Pro are not cleaved (Asp forms a cyclic anhydride that does not react with ammonium thiocyanate); this limitation can be circumvented by modifying both carboxyl groups of Asp with a carbodiimide [35] and enzymatically removing the protecting group from the α-carboxyl [44]).

Other modifications include the use of TFA and acetyl chloride instead of acetic anhydride [48] to ensure that the reaction proceeds primarily via mixed anhydride formation rather than oxazolinone [98]. In addition, it was proposed to use thiocyanic acid itself rather than thiocyanates in the reaction [48]. Under these conditions, successful amino acid sequencing of peptides was achieved, including those possessing a C-terminal Pro. Carrying out the reaction with The addition of trifluoroacetic anhydride [96] made it possible to determine 14 amino acids of Papain and 10 amino acids of Ribonuclease [97].

When performing the process in solution, Sephadex Chromatography is typically used to separate excess Reagents from the peptidyl thiohydantoin, and the thiohydantoin from the shortened peptide. These separations and the lyophilization of Column fractions are time-consuming. Solid-phase analysis eliminates many of these problems, but an unfortunate choice (or preliminary synthesis) of the activated support and the difficulties involved in attaching the peptide to the support drastically reduce the chances of a successful determination. A detailed Description of the techniques is given below.

18.4.1.1. Thiocyanate Cleavage of Peptides. Formation of Peptidyl Thiohydantoins. Method 1 [83]. Dissolve the peptide in 0.5 mL of 50% acetic acid. To the peptide solution, add slowly with stirring a freshly prepared solution of ammonium thiocyanate (100 mg), acetic anhydride (4.0 mL), and glacial acetic acid (1.0 mL). Heat at 50 °C for 6 h, add another 100 mg of ammonium thiocyanate with stirring, and heat the reaction mixture at 50 °C for an additional 18 h. Then add Water (3.0 mL) and let the solution stand for a few minutes to hydrolyze the excess acetic anhydride.

Method 2 [87]. Treat the peptide with 1.0 mL of an acetylating mixture: acetic acid – acetic anhydride – pyridine (2–10:1) at 50 °C for 30 min, then add 1.0 mL of an ammonium thiocyanate solution

in acetic acid (20 mg/mL), and heat the reaction mixture at 50 °C with vigorous stirring for 30 min.

Method 3 [48]. Dissolve the peptide in a mixture of TFA (10 μL) and acetyl chloride (200 μL), and incubate the reaction mixture at 30 °C for 15 min. Add a solution of 3% thiocyanic acid in dioxane (200 μL) and keep the reaction mixture at 30 °C for another 60 min. Once the peptidyl thiohydantoin is formed, the solvent can be removed in vacuo.

Isolation of Peptidyl Thiohydantoin.

Method 1 [83]. Separate the peptidyl thiohydantoin from excess reagents by Desalting on a Sephadex G-25 column (20 × 500 mm) in 50% acetic acid. Collect 5 mL fractions, detecting the peptidyl thiohydantoin by UV absorption; pool the desired fractions.

Method 2 [87]. Isolation is carried out without chromatographic Separation. Cleavage is performed directly in the reaction mixture after evaporating it to dryness.

Cleavage of Peptidyl Thiohydantoins.

Method 1 [83]. Pool the fractions containing the peptidyl thiohydantoin; evaporate the solvent in vacuo. Dissolve the dry residue in 0.5 mL of 0.1 M acetohydroxamic acid in 50% pyridine. Heat the solution at 50 °C for 2 h and evaporate to dryness. Dissolve the dry residue in 3 mL of 50% acetic acid, and separate the cleaved C-terminal amino acid thiohydantoin from the shortened peptide by column chromatography on Sephadex G-25 (20 × 500 mm).

Method 2 [87]. Evaporate the solution to dryness without separation. Dissolve the residue in water (3 mL), and cleave the thiohydantoin by adding 2 g of a cation exchanger (Dowex 50 WX8) to the reaction mixture and stirring at 50 °C for 30 min.

Method 3 [48]. Cleave the thiohydantoin with 0.5 M triethylamine.

18.4.1.2. Thiocyanate Cleavage of Proteins. Since proteins are sparingly soluble in the acetic acid – acetic anhydride mixture, the use of hexafluoroacetone trihydrate [18] and trifluoroacetic anhydride [96] has been proposed.

Formation of Protein Thiohydantoin.

Method 1 [18]. Dissolve the protein (0.015–1.0 μmol) in a mixture of hexafluoroacetone trihydrate (1.0 mL) and water (0.35 mL). Add dropwise with stirring a freshly prepared, clear solution of ammonium thiocyanate (100 mg) in a mixture of hexafluoroacetone trihydrate (1.0 mL) and acetic anhydride (4.5 mL).

Heat the mixture at 50 °C for 2 h, then add another 100 mg of ammonium thiocyanate and heat for an additional 18 h. To destroy excess acetic anhydride, add water (3.0 mL). Separate the protein thiohydantoin from by-products on Sephadex G-25.

Cleavage of Protein Thiohydantoin.

Method 1 [18]. Pool the eluate fractions containing the protein thiohydantoin; remove the solvent in vacuo. Dissolve the residue in 1.0 mL of 12 M HCl and let stand at room temperature for 30 min. Remove the Hydrochloric acid by rapid evaporation in vacuo. Dissolve the residue in 50% acetic acid, and separate the cleaved thiohydantoin from the shortened protein by Gel filtration as described above. Immediately evaporate the solvent, add a small volume of methanol, and identify the thiohydantoin without delay (since thiohydantoins are unstable in light and air) by TLC.

18.4.1.3. Identification of Thiohydantoins. Following the cleavage of thiohydantoins from small peptides, Amino acid analysis of the shortened peptide can be performed to determine the C-terminal amino acid from the difference in the Amino Acid Composition of the peptides before and after cleavage. For proteins, direct identification of the cleaved thiohydantoins should be carried out, for example, by TLC on silica gel plates impregnated with a fluorescent indicator. Chromatography is performed either in the heptane – n-butanol – 99% formic acid system (95:65:30) or in the chloroform – 95% ethanol – glacial acetic acid system (100:50:15) [18, 83, 84].

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FIG. 18.10. Two-dimensional TLC of amino acid thiohydantoins on polyamide (100×100 mm plates). Chromatography in the first direction: glacial acetic acid — water (7:13); In the second direction: chloroform — 95% ethanol — glacial acetic acid mixture (20:20:3) containing a 2,5-bis(5-tert-butyl-benzoxazolyl-2)thiophene fluorescent indicator (0.025%). Under UV illumination (254 nm), the thiohydantoins appear as dark spots against a pale blue fluorescent Background [70].

Two-dimensional TLC on polyamide plates (100×100 mm) is more efficient, using an acetic acid — water system (7:13) in the first direction and a chloroform — 95% ethanol — glacial acetic acid mixture (20:10:3) containing 0.025% 2,5-bis(5-tert-butylbenzoxazolyl-2)thiophene in the second direction [70]. Upon UV irradiation (254 nm), amino acid thiohydantoins are visualized as dark spots against a pale blue fluorescent background (Fig. 18.10).

Amino acid thiohydantoins can be identified by GLC following trimethylsilylation [23, 72], by mass spectrometry [19, 72, 86, 87], and also following Hydrolysis of the thiohydantoins to free amino acids using Ion-exchange chromatography [84] and GLC [70].